Methods and compositions for mycoplasma pneumoniae exotoxins

ABSTRACT

The present invention provides a  Mycoplasma pneumoniae  community acquired respiratory distress syndrome (CARDS) toxin, biologically active fragments/domains of the CARDS toxin, antibodies to the CARDS toxin and nucleic acids encoding the CARDS toxin. Also provided are methods of diagnosing, treating and/or preventing infection by  Mycoplasma pneumoniae  using the compositions provided herein.

RELATED APPLICATIONS

This application is a divisional application of, and claims priority to,U.S. application Ser. No. 10/573,909, having a filing date of Jan. 8,2007, which application is allowed, and which claims the benefit, under35 U.S.C. §119(e), of U.S. Provisional Application Ser. No. 60/508,607,filed Oct. 3, 2003, the entire contents of each of which areincorporated herein by reference.

STATEMENT OF GOVERNMENT SUPPORT

Research related to this invention was supported, at least in part, byU.S. Government Grant No. AI45737 awarded by the NIAID. The Governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to Mycoplasma pneumoniae exotoxins,antibodies thereto, and their use in diagnostic and therapeutic methods.

2. Background Art

Mycoplasma pneumoniae is one of the most well recognized pathogens ofthe human respiratory tract. The importance of Mycoplasma pneumoniae asa cause of human respiratory disease has been well documented byepidemiological studies in various settings and in many countries. M.pneumoniae is the etiologic agent of primary atypical pneumonia and isalso responsible for many respiratory tract infections, such astracheobronchitis, bronchiolitis, pharyngitis and croup, especially inolder children and young adults and in elderly populations. It accountsfor 20-30% of all pneumonias and also is linked to asthma and chronicobstructive pulmonary disease. Furthermore, M. pneumoniae candisseminate to other organ sites and cause gastrointestinal,hematologic, neurologic, dermatologic, musculoskeletal andcardiovascular pathologies. This secondary involvement by M. pneumoniaeleads to a spectrum of complicated extrapulmonary sequelae, includingarthritis, pericarditis and central nervous system disorders, whichattests to the significance of M. pneumoniae in human disease. Althoughantibiotic therapy appears to be relatively effective in controllingmycoplasma pneumonia, the bacteria continue to persist.

At present, no known virulence determinants of M. pneumoniae have beenfunctionally identified and linked to the wide range of pathologiesassociated with M. pneumoniae mediated diseases. Furthermore, there areno specific and standardized diagnostic tests available for reliable andrapid detection of M. pneumoniae infection, or effective vaccines tocontrol M. pneumoniae infection.

The present invention overcomes previous shortcomings in the art byproviding a Mycoplasma pneumoniae polypeptide and biologically activefragments thereof, known as community acquired respiratory distresssyndrome (CARDS) toxin, as well as nucleic acids encoding thispolypeptide and its fragments and antibodies specific thereto. Thesecompositions are used, for example, in methods of diagnosing, treatingand preventing infection by M. pneumoniae.

SOME SEQUENCES OF THIS INVENTION

Reference amino acid sequence M129/B9 (reference strain): (SEQ ID NO: 1)MPNPVRFVYR VDLRSPEEIF EHGFSTLGDV RNFFEHILST NFGRSYFIST SETPTAAIRFFGSWLREYVP EHPRRAYLYE IRADQHFYNA RATGENLLDL MRQRQVVFDS GDREMAQMGIRALRTSFAYQ REWFTDGPIA AANVRSAWLV DAVPVEPGHA HHPAGRVVET TRINEPEMHNPHYQELQTQA NDQPWLPTPG IATPVHLSIP QAASVADVSE GTSASLSFAC PDWSPPSSNGENPLDKCIAE KIDNYNLQSL PQYASSVKEL EDTPVYLRGI KTQKTFMLQA DPQNNNVFLVEVNPKQKSSF PQTIFFWDVY QRICLKDLTG AQISLSLTAF TTQYAGQLKV HLSVSAVNAVNQKWKMTPQD IAITQFRVSS ELLGQTENGL FWNTKSGGSQ HDLYVCPLKN PPSDLEELQIIVDECTTHAQ FVTMRAASTF FVDVQLGWYW RGYYYTPQLS GWSYQMKTPD GQIFYDLKTSKIFFVQDNQN VFFLHNKLNK QTGYSWDWVE WLKHDMNEDK DENFKWYFSR DDLTIPSVEGLNFRHIRCYA DNQQLKVIIS GSRWGGWYST YDKVESNVED KILVKDGFDR F S1 (clinicalstrain) amino acid sequence: (SEQ ID NO: 2)MPNPVRFVYRVDLRSPEEIFEHGESTLGDVRNFFEHIPSTNEGRSYFISTSETPTAAIRFFGSWLREYVPEHPRRAYLYEIRADQHFYNARATGENLLDLMRQRQVVEDSGDREMAQMGIRALRTSFAYQREWFTDGPIAAANVRSAWLVDAVPVERGHAHHPAGRVVETTRINEPEMHNPHYQELQTQANDQPWLPTPGIATPVHLSIPQAASVADVSEGTSASLSFACPDWSPPSSNGENPLDKCIAEKIDNYNLQSLPQYASSVKELEDTPVYLRGIKTQKTFMLQADPQNNNVFLVEVNPKQKSPFPQTIFFWDVYQRICLKDLTGAQISLSLTAFTTQYAGQLKVHLSVSAVNAVNQKWKMTPQDSAITQFRVSSELLGQTENGLSWNTKSGGSQHDLYVCPLKNPPSDLEELQIIVDECTTHAQFVTMRAASTFFVDVQLGWYWRGYYYTPQLSGWSYQMKTPDGQIFYDLKTSKIFFVQDNQNVFFLHNKLNKQTGYSWDWVEWLKHDMNEDKDENFKWYFSRDDLTIPSVEGLNFRHIRCYADNQQLKVIISGSRWGGWYSTYDKVESNVEDKILVKDGFDRF JL (clinical strain)amino acid sequence: (SEQ ID NO: 3)MPNPVRFVYRVDLRSPEEIFEHGFSTLGDVRNFFEHILSTNFGRSYFISTSETPTAAIRFFGSWLREYVPFHPRRAYLYEIRADQHFYNARATGENLLDLMRQRQVVFDSGDREMAQMGIRALRTSFAYQREWFTDGPIAAANVRSAWLVDAVPVEPGHAHHPAGRVVETTRINEPEMHNPHYQELQTQANDQPWLPTPGIATPVHLSIPQAASVADVSEGTSASLSFACPDWSPPSSNGENPLDKCIAEKIDNYNLQSLPQYASSVKELEDTPVYLRGIKTQKTFMLQADPQNNNVFLVEVNPKQKSSFPQTIFFWDVYQRICLKDLTGAQISLSLTAFTTQYAGQLKVHLSVSAVNAVNQKWKMTPQDSAITQFRVSSELLGQTENGLFWNTKSGGSQHDLYVCPLKNPPSDLEELQIIVDECTTHAQFVTMRAASTFFVDVQLGWYWRGYYYTPQLSGWSYQMKTPDGQIFYDLKTSKIFFVQDNQNVFFLHNKLNKQTGYSWDWVEWLKHDMNEDKDENFKWYFSRDDLTIPSVEGLNFRHIRCYADNQQLKVIISGSRWGGWYSTYDKVESNVEDKILVKDGFDRF RJL1 (clinicalstrain) amino acid sequence: (SEQ ID NO: 4)MPNPVRFVYRVDLRSPEEIFEHGFSTLGDVRNFFEHILSTNFGRSYFISTSETPTAAIRFFGSWLREYVPEMPRRAYLYEIRADQHFYNARATGENLLDLMRQRQVVFDSGDREMAQMGIRALRTSFAYQREWFTDGPIAAANVRSAWLVDAVPVEPGHAHHPAGRVVETTRINEPEMHNPHYQELQTQANDQPWLPTPGIATPVHLSIPQAASVADVSEGTSASLSFACPDWSPPSSNGENPLDKCIAEKIDNYNLQSLPQYASSVKELEDTPVYLRGIKTQKTFMLQADPQNNNVFLVEVNPKQKSSFPQTIFFWDVYQRICLKDLTGAQISLSLTAFTTQYAGQLKVHLSVSAVNAVNQKWKMTPQDSAITQFRVSSELLGQTENGLFRNTKSGGSQHDLYVCPLKNPPSDLEELQIIVDECTTHAQFVTMRAASTFFVDVQLGWYWRGYYYTPQLSGWSYQMKTPDGQIFYDLKTSKIFFVQDNQNVFFLHNKLNKQTGYSWDWVEWLKHDMNEDKDENFKWYFSRDDLTIPSVEGLNFRHIRCYADNQQLKVIISGSRWGGWYSTYDKVESNVEDKILVKDGFDRF L2 (clinical strain)amino acid sequence: (SEQ ID NO: 5)MPNPVRFVYRVDLRSPEEIFEHGFSTLGDVRNFFEHILSTNFGRSYFISTSETPTAAIRFFGSWLREYVPEHPRRAYLYEIRADQHFYNARATGENLLDLMRQRQVVFDSGDREMAQMGIRALRTSFAYQREWFTDGPIAAANVRSAWLVDAVPVEPGHAHHPAGRVVETTRINEPEMHNPHYQELQTQANDQPWLPTPGIATPVHLSIPQAASVADVSEGTSASLSFACPDWSPPSSNGENPLGKCIAEKIDNYNLQSLPQYASSVKELEDTPVYLRGIKTQKTFMLQADPQNNNVFLVEVNPKQKSSFPQTIFFWDVYQRICLKDLTGAQISLSLTAFTTQYAGQLKVHLSVSAVNAVNQKWKMTPQDSAITQFRVSSELLGQTENGLFWNTKSGGSQHDLYVCPLKNPPSDLEELQIIVDECTTHAQFVTMRAASTFFVDVQLGWYWRGYYYTPQLSGWSYQMKTPDGQIFYDLKTSKIFFVQDNQNVFFLHNKLNKQTGYSWDWVEWLKHDMNEDKDENFKWYFSRDDLTIPSVEGLNFRHIRCYADNQQLKVIISGSRWGGWYSTYDKVESNVEDKILVKDGFDRF Composite amino acidsequence: (SEQ ID NO: 6) MPNPVRFVYR VDLRSPEEIF EHGFSTLGDV RNFFEHIPSTNFGRSYFIST SETPTAAIRF FGSWLREYVP EHPRRAYLYE IRADQHFYNA RATGENLLDLMRQRQVVFDS GDREMAQMGI RALRTSFAYQ REWFTDGPIA AANVRSAWLV DAVPVEPGHAHHPAGRVVET TRINEPEMHN PHYQELQTQA NDQPWLPTPG IATPVHLSIP QAASVADVSEGTSASLSFAC PDWSPPSSNG ENPLGKCIAE KIDNYNLQSL PQYASSVKEL EDTPVYLRGIKTQKTFMLQA DPQNNNVFLV EVNPKQKPSF PQTIFFWDVY QRICLKDLTG AQISLSLTAFTTQYAGQLKV HLSVSAVNAV NQKWKMTPQD SAITQFRVSS ELLGQTENGL SRNTKSGGSQHDLYVCPLKN PPSDLEELQI IVDECTTHAQ FVTMRAASTF FVDVQLGWYW RGYYYTPQLSGWSYQMKTPD GQIFYDLKTS KIFFVQDNQN VFFLHNKLNK QTGYSWDWVE WLKHDMNEDKDENFKWYFSR DDLTIPSVEG LNFRHIRCYA DNQQLKVIIS GSRWGGWYST YDKVESNVEDKILVKDGFDR F Reference nucleotide sequence M129/B9 (contains tga's thatneed to be changed to tgg before expression in E. coli) (SEQ ID NO: 7)tttttaattt gtaaaatttc attttttaaa aatgccaaat cctgttagat ttgtttaccgtgttgatttg agaagccctg aagaaatttt tgaacatggc ttttcaactt taggtgatgtgagaaatttc tttgaacaca ttctctccac taattttggt agaagctatt ttatttccacttcagaaaca cccacagcag ctattcgctt ctttggtagc tggttacggg aatatgtaccagagcacccc agaagggctt acttatatga aattcgtgcc gaccaacact tttacaatgcccgcgccact ggggagaact tgttagattt aatgcgtcaa agacaagtag tatttgactctggtgatcga gaaatggcac aaatgggaat tagagcttta cgcacttcct ttgcgtatcaacgtgaatgg tttaccgatg gtccaattgc agcagctaat gtccgtagtg cttgactagtagatgctgtt cccgttgaac ctggtcatgc tcaccacccg gctggtcgtg ttgtagagactactagaatt aatgaaccgg aaatgcacaa ccctcattat caagagctgc aaacccaagccaatgatcaa ccatgattgc caacaccagg aatagctact cctgtacatt tatcaattccccaagcagct tccgttgctg atgtttcgga aggtacttcc gcttcgctat cgtttgcgtgccctgattga agtccacctt ctagtaatgg tgaaaatccg ctagacaaat gcattgcggaaaagattgat aactataacc tacaatcctt accacagtac gctagcagtg taaaggaactggaagataca ccagtatacc taaggggaat taaaacgcaa aaaaccttta tgttacaagcagatccgcaa aataacaatg tctttttggt cgaagtaaac cccaaacaaa agtccagctttccccaaacc atcttctttt gggatgttta tcaacgaatt tgtctcaagg atttaactggtgcacaaatc agtctttcgc ttactgcctt tactactcag tatgctggtc agctcaaagtgcaccttagt gttagcgcgg ttaatgccgt gaaccaaaag tgaaaaatga caccgcaagacattgcaata actcagtttc gggtctcctc tgaactgtta ggtcaaactg aaaatggcttgttctgaaat accaagagtg gtggttcaca acacgatttg tatgtatgtc ctttgaaaaatccacctagt gatttggaag aattacaaat aattgttgat gaatgtacta cccatgcgcagtttgttact atgcgtgcag ctagcacctt ctttgttgat gttcagctag gctggtattgaaggggttat tactataccc cacaattaag tggttgatct tatcagatga aaacaccagatggacagata ttctatgatc taaaaacttc gaaaatcttc tttgtccagg acaaccaaaacgtgttcttt ctccataata aactcaacaa acaaactggt tacagctggg attgagtagaatggctaaaa catgacatga atgaggacaa agacgaaaac tttaaatggt acttttcgcgtgatgacctt accattcctt ccgttgaagg gcttaacttc cgccacattc gctgttacgctgacaaccag cagttaaagg tgatcataag cggttcacgt tggggcggtt ggtactccacttacgataaa gttgaaagta atgtcgaaga taagattttg gtcaaagatg gttttgatcgcttttagcga ttaagcttta acgtcactgt tttgctctaa tgttagaagc aaagatcttg S1Nucleotide sequence with each tga changed to tgg for expression in E.coli (SEQ ID NO: 8) atgccaaatc ctgttagatt tgtttaccgt gttgatttgagaagccctga agaaattttt   60 gaacatggct tttcaacttt aggtgatgtg agaaatttctttgaacacat tccctccact  120 aattttggta gaagctattt tatttccact tcagaaacacccacagcagc tattcgcttc  180 tttggtagct ggttacggga atatgtacca gagcaccccagaagggctta cttatatgaa  240 attcgtgccg accaacactt ttacaatgcc cgcgccactggggagaactt gttagattta  300 atgcgtcaaa gacaagtagt atttgactct ggtgatcgagaaatggcaca aatgggaatt  360 agagctttac gcacttcctt tgcgtatcaa cgtgaatggtttaccgatgg tccaattgca  420 gcagctaatg tccgtagtgc ttggctagta gatgctgttcccgttgaacc tggtcatgct  480 caccacccgg ctggtcgtgt tgtagagact actagaattaatgaaccgga aatgcacaac  540 cctcattatc aagagctgca aacccaagcc aatgatcaaccatggttgcc aacaccagga  600 atagctactc ctgtacattt atcaattccc caagcagcttccgttgctga tgtttcggaa  660 ggtacttccg cttcgctatc gtttgcgtgc cctgattggagtccaccttc tagtaatggt  720 gaaaatccgc tagacaaatg cattgcggaa aagattgataactataacct acaatcctta  780 ccacagtacg ctagcagtgt aaaggaactg gaagatacaccagtatacct aaggggaatt  840 aaaacgcaaa aaacctttat gttacaagca gatccgcaaaataacaatgt ctttttggtc  900 gaagtaaacc ccaaacaaaa gcccagcttt ccccaaaccatcttcttttg ggatgtttat  960 caacgaattt gtctcaagga tttaactggt gcacaaatcagtctttcgct tactgccttt 1020 actactcagt atgctggtca gctcaaagtg caccttagtgttagcgcggt taatgccgtg 1080 aaccaaaagt ggaaaatgac accgcaagac agtgcaataactcagtttcg ggtctcctct 1140 gaactgttag gtcaaactga aaatggcttg tcctggaataccaagagtgg tggttcacaa 1200 cacgatttgt atgtatgtcc tttgaaaaat ccacctagtgatttggaaga attacaaata 1260 attgttgatg aatgtactac ccatgcgcag tttgttactatgcgtgcagc tagcaccttc 1320 tttgttgatg ttcagctagg ctggtattgg aggggttattactatacccc acaattaagt 1380 ggttggtctt atcagatgaa aacaccagat ggacagatattctatgatct aaaaacttcg 1440 aaaatcttct ttgtccagga caaccaaaac gtgttctttctccataataa actcaacaaa 1500 caaactggtt acagctggga ttgggtagaa tggctaaaacatgacatgaa tgaggacaaa 1560 gacgaaaact ttaaatggta cttttcgcgt gatgaccttaccattccttc cgttgaaggg 1620 cttaacttcc gccacattcg ctgttacgct gacaaccagcagttaaaggt gatcataagc 1680 ggttcacgtt ggggcggttg gtactccact tacgataaagttgaaagtaa tgtcgaagat 1740 aagattttgg tcaaagatgg ttttgatcgc ttt 1773 L2nucleotide sequence with each tga changed to tgg for expression in E.coli (SEQ ID NO: 9) atgccaaatc ctgttagatt tgtttaccgt gttgatttgagaagccctga agaaattttt   60 gaacatggct tttcaacttt aggtgatgtg agaaatttctttgaacacat tctctccact  120 aattttggta gaagctattt tatttccact tcagaaacacccacagcagc tattcgcttc  180 tttggtagct ggttacggga atatgtacca gagcaccccagaagggctta cttatatgaa  240 attcgtgccg accaacactt ttacaatgcc cgcgccactggggagaactt gttagattta  300 atgcgtcaaa gacaagtagt atttgactct ggtgatcgagaaatggcaca aatgggaatt  360 agagctttac gcacttcctt tgcgtatcaa cgtgaatggtttaccgatgg tccaattgca  420 gcagctaatg tccgtagtgc ttggctagta gatgctgttcccgttgaacc tggtcatgct  480 caccacccgg ctggtcgtgt tgtagagact actagaattaatgaaccgga aatgcacaac  540 cctcattatc aagagctgca aacccaagcc aatgatcaaccatggttgcc aacaccagga  600 atagctactc ctgtacattt atcaattccc caagcagcttccgttgctga tgtttcggaa  660 ggtacttccg cttcgctatc gtttgcgtgc cctgattggagtccaccttc tagtaatggt  720 gaaaatccgc taggcaaatg cattgcggaa aagattgataactataacct acaatcctta  780 ccacagtacg ctagcagtgt aaaggaactg gaagatacaccagtatacct aaggggaatt  840 aaaacgcaaa aaacctttat gttacaagca gatccgcaaaataacaatgt ctttttggtc  900 gaagtaaacc ccaaacaaaa gtccagcttt ccccaaaccatcttcttttg ggatgtttat  960 caacgaattt gtctcaagga tttaactggt gcacaaatcagtctttcgct tactgccttt 1020 actactcagt atgctggtca gctcaaagtg caccttagtgttagcgcggt taatgccgtg 1080 aaccaaaagt ggaaaatgac accgcaagac agtgcaataactcagtttcg ggtctcctct 1140 gaactgttag gtcaaactga aaatggcttg ttctggaataccaagagtgg tggttcacaa 1200 cacgatttgt atgtatgtcc tttgaaaaat ccacctagtgatttggaaga attacaaata 1260 attgttgatg aatgtactac ccatgcgcag tttgttactatgcgtgcagc tagcaccttc 1320 tttgttgatg ttcagctagg ctggtattgg aggggttattactatacccc acaattaagt 1380 ggttggtctt atcagatgaa aacaccagat ggacagatattctatgatct aaaaacttcg 1440 aaaatcttct ttgtccagga caaccaaaac gtgttctttctccataataa actcaacaaa 1500 caaactggtt acagctggga ttgggtagaa tggctaaaacatgacatgaa tgaggacaaa 1560 gacgaaaact ttaaatggta cttttcgcgt gatgaccttaccattccttc cgttgaaggg 1620 cttaacttcc gccacattcg ctgttacgct gacaaccagcagttaaaggt gatcataagc 1680 ggttcacgtt ggggcggttg gtactccact tacgataaagttgaaagtaa tgtcgaagat 1740 aagattttgg tcaaagatgg ttttgatcgc ttt 1773 JLnucleotide sequence with each tga changed to tgg for expression in E.coli (SEQ ID NO: 10) atgccaaatc ctgttagatt tgtttaccgt gttgatttgagaagccctga agaaattttt   60 gaacatggct tttcaacttt aggtgatgtg agaaatttctttgaacacat tctctccact  120 aattttggta gaagctattt tatttccact tcagaaacacccacagcagc tattcgcttc  180 tttggtagct ggttacggga atatgtacca gagcaccccagaagggctta cttatatgaa  240 attcgtgccg accaacactt ttacaatgcc cgcgccactggggagaactt gttagattta  300 atgcgtcaaa gacaagtagt atttgactct ggtgatcgagaaatggcaca aatgggaatt  360 agagctttac gcacttcctt tgcgtatcaa cgtgaatggtttaccgatgg tccaattgca  420 gcagctaatg tccgtagtgc ttggctagta gatgctgttcccgttgaacc tggtcatgct  480 caccacccgg ctggtcgtgt tgtagagact actagaattaatgaaccgga aatgcacaac  540 cctcattatc aagagctgca aacccaagcc aatgatcaaccatggttgcc aacaccagga  600 atagctactc ctgtacattt atcaattccc caagcagcttccgttgctga tgtttcggaa  660 ggtacttccg cttcgctatc gtttgcgtgc cctgattggagtccaccttc tagtaatggt  720 gaaaatccgc tagacaaatg cattgcggaa aagattgataactataacct acaatcctta  780 ccacagtacg ctagcagtgt aaaggaactg gaagatacaccagtatacct aaggggaatt  840 aaaacgcaaa aaacctttat gttacaagca gatccgcaaaataacaatgt ctttttggtc  900 gaagtaaacc ccaaacaaaa gtccagcttt ccccaaaccatcttcttttg ggatgtttat  960 caacgaattt gtctcaagga tttaactggt gcacaaatcagtctttcgct tactgccttt 1020 actactcagt atgctggtca gctcaaagtg caccttagtgttagcgcggt taatgccgtg 1080 aaccaaaagt ggaaaatgac accgcaagac agtgcaataactcagtttcg ggtctcctct 1140 gaactgttag gtcaaactga aaatggcttg ttctggaataccaagagtgg tggttcacaa 1200 cacgatttgt atgtatgtcc tttgaaaaat ccacctagtgatttggaaga attacaaata 1260 attgttgatg aatgtactac ccatgcgcag tttgttactatgcgtgcagc tagcaccttc 1320 tttgttgatg ttcagctagg ctggtattgg aggggttattactatacccc acaattaagt 1380 ggttggtctt atcagatgaa aacaccagat ggacagatattctatgatct aaaaacttcg 1440 aaaatcttct ttgtccagga caaccaaaac gtgttctttctccataataa actcaacaaa 1500 caaactggtt acagctggga ttgggtagaa tggctaaaacatgacatgaa tgaggacaaa 1560 gacgaaaact ttaaatggta cttttcgcgt gatgaccttaccattccttc cgttgaaggg 1620 cttaacttcc gccacattcg ctgttacgct gacaaccagcagttaaaggt gatcataagc 1680 ggttcacgtt ggggcggttg gtactccact tacgataaagttgaaagtaa tgtcgaagat 1740 aagattttgg tcaaagatgg ttttgatcgc ttt 1773RJL1 nucleotide sequence with each tga changed to tgg for expression inE. coli (SEQ ID NO: 11) atgccaaatc ctgttagatt tgtttaccgt gttgatttgagaagccctga agaaattttt   60 gaacatggct tttcaacttt aggtgatgtg agaaatttctttgaacacat tctctccact  120 aattttggta gaagctattt tatttccact tcagaaacacccacagcagc tattcgcttc  180 tttggtagct ggttacggga atatgtacca gagcaccccagaagggctta cttatatgaa  240 attcgtgccg accaacactt ttacaatgcc cgcgccactggggagaactt gttagattta  300 atgcgtcaaa gacaagtagt atttgactct ggtgatcgagaaatggcaca aatgggaatt  360 agagctttac gcacttcctt tgcgtatcaa cgtgaatggtttaccgatgg tccaattgca  420 gcagctaatg tccgtagtgc ttggctagta gatgctgttcccgttgaacc tggtcatgct  480 caccacccgg ctggtcgtgt tgtagagact actagaattaatgaaccgga aatgcacaac  540 cctcattatc aagagctgca aacccaagcc aatgatcaaccatggttgcc aacaccagga  600 atagctactc ctgtacattt atcaattccc caagcagcttccgttgctga tgtttcggaa  660 ggtacttccg cttcgctatc gtttgcgtgc cctgattggagtccaccttc tagtaatggt  720 gaaaatccgc tagacaaatg cattgcggaa aagattgataactataacct acaatcctta  780 ccacagtacg ctagcagtgt aaaggaactg gaagatacaccagtatacct aaggggaatt  840 aaaacgcaaa aaacctttat gttacaagca gatccgcaaaataacaatgt ctttttggtc  900 gaagtaaacc ccaaacaaaa gtccagcttt ccccaaaccatcttcttttg ggatgtttat  960 caacgaattt gtctcaagga tttaactggt gcacaaatcagtctttcgct tactgccttt 1020 actactcagt atgctggtca gctcaaagtg caccttagtgttagcgcggt taatgccgtg 1080 aaccaaaagt ggaaaatgac accgcaagac agtgcaataactcagtttcg ggtctcctct 1140 gaactgttag gtcaaactga aaatggcttg ttccggaataccaagagtgg tggttcacaa 1200 cacgatttgt atgtatgtcc tttgaaaaat ccacctagtgatttggaaga attacaaata 1260 attgttgatg aatgtactac ccatgcgcag tttgttactatgcgtgcagc tagcaccttc 1320 tttgttgatg ttcagctagg ctggtattgg aggggttattactatacccc acaattaagt 1380 ggttggtctt atcagatgaa aacaccagat ggacagatattctatgatct aaaaacttcg 1440 aaaatcttct ttgtccagga caaccaaaac gtgttctttctccataataa actcaacaaa 1500 caaactggtt acagctggga ttgggtagaa tggctaaaacatgacatgaa tgaggacaaa 1560 gacgaaaact ttaaatggta cttttcgcgt gatgaccttaccattccttc cgttgaaggg 1620 cttaacttcc gccacattcg ctgttacgct gacaaccagcagttaaaggt gatcataagc 1680 ggttcacgtt ggggcggttg gtactccact tacgataaagttgaaagtaa tgtcgaagat 1740 aagattttgg tcaaagatgg ttttgatcgc ttt 1773

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an immunoblot that demonstrates both production of theCARDS toxin and anti-CARDS antibodies in three patients during infectionwith Mycoplasma pneumoniae.

FIG. 2 shows ADP-ribosylation of G proteins in HEp-2 cells followingincubation with CARDS protein. Lane 1: HEp-2 cells in medium alonefollowed by preparation of cell free extract and addition of CARDSprotein. Lane 2: HEp-2 cells pretreated with CARDS protein, followed bypreparation of cell free extract and addition of CARDS protein. Themarked reduction in ADP-ribosylation of specific proteins in the CARDSprotein-pretreated cells is indicated by arrows. Also, ADP-ribosylationof other Hep-2 cell proteins is diminished (lane 2).

FIG. 3 shows an ELISA and an immunoblot employing rD1 as antigen thatdemonstrates production of anti-CARDS antibodies in sequential serumsamples of two patients infected with Mycoplasma pneumoniae.

SUMMARY OF THE INVENTION

The present invention provides Mycoplasma pneumoniae exotoxin (CARDStoxin) from subjects infected with Mycoplasma pneumoniae. In particular,the present invention provides a polypeptide comprising, consistingessentially of, and/or consisting of the amino acid sequence of SEQ IDNO:2 (S1 isolate), a polypeptide comprising, consisting essentially of,and/or consisting of the amino acid sequence of SEQ ID NO:3 (JLisolate), a polypeptide comprising, consisting essentially of, and/orconsisting of the amino acid sequence of SEQ ID NO:4 (RJL1 isolate), apolypeptide comprising, consisting essentially of, and/or consisting ofthe amino acid sequence of SEQ ID NO:5 (L2 isolate), a polypeptidecomprising, consisting essentially of, and/or consisting of the aminoacid sequence of SEQ ID NO:1 (reference sequence), and/or a polypeptidecomprising, consisting essentially of, and/or consisting of the aminoacid sequence of SEQ ID NO:6 (composite sequence), either individuallyor in any combination.

The present invention further provides biologically active fragments ofthe polypeptides of this invention, as well as antibodies thatspecifically bind the polypeptides and/or fragments of the polypeptidesof this invention.

Further provided are nucleotide sequences that encode the polypeptidesand fragments of this invention. In particular, the present inventionprovides an isolated nucleic acid comprising, consisting essentially of,and/or consisting of the nucleotide sequence of SEQ ID NO:8 (S1isolate), an isolated nucleic acid comprising, consisting essentiallyof, and/or consisting of the nucleotide sequence of SEQ ID NO:10 (JLisolate), an isolated nucleic acid comprising, consisting essentiallyof, and/or consisting of the nucleotide sequence of SEQ ID NO:11 (RJL1isolate), an isolated nucleic acid comprising, consisting essentiallyof, and/or consisting of the nucleotides sequence of SEQ ID NO:9 (L2isolate), an isolated nucleic acid comprising, consisting essentiallyof, and/or consisting of the nucleotides sequence of SEQ ID NO:7(reference sequence), and/or an isolated nucleic acid comprising,consisting essentially of, and/or consisting of the nucleotide sequenceof SEQ ID NO:76 (composite sequence), either individually or in anycombination.

Additionally provided is a nucleic acid comprising, consistingessentially of and/or consisting of a nucleotide sequence that encodesan amino acid sequence comprising, consisting essentially of and/orconsisting of the amino acid sequence or a biologically active fragmentof the amino acid sequence of SEQ ID NO:2 (S1 isolate), a nucleic acidcomprising, consisting essentially of, and/or consisting of a nucleotidesequence that encodes an amino acid sequence comprising, consistingessentially of, and/or consisting of the amino acid sequence or abiologically active fragment of the amino acid sequence of SEQ ID NO:3(JL isolate), a nucleic acid comprising, consisting essentially of,and/or consisting of a nucleotide sequence that encodes an amino acidsequence comprising, consisting essentially of, and/or consisting of theamino acid sequence or a biologically active fragment of the amino acidsequence of SEQ ID NO:4 (RJL1 isolate), a nucleic acid comprising,consisting essentially of and/or consisting of a nucleotide sequencethat encodes an amino acid comprising, consisting essentially of, and/orconsisting of the amino acid sequence or a biologically active fragmentof an amino acid sequence of SEQ ID NO:5 (L2) isolate, a nucleic acidcomprising, consisting essentially of, and/or consisting of a nucleotidesequence that encodes an amino acid comprising, consisting essentiallyof, and/or consisting of the amino acid sequence or a biologicallyactive fragment of an amino acid sequence of SEQ ID NO:1 (referencesequence) isolate, and/or a nucleic acid comprising, consistingessentially of, and/or consisting of a nucleotide sequence encoding anamino acid sequence comprising, consisting essentially of, and/orconsisting of the amino acid sequence or a biologically active fragmentof the amino acid sequence of SEQ ID NO:6 (composite sequence). Furtherprovided herein is a nucleic acid that is the complement of each and anyof the nucleic acids of this invention.

Also provided herein are probes and primers for the detection and/oramplification of the nucleic acids of this invention, including

(SEQ ID NO: 12; Primer 1) TTTTTACATATGCCAAATCCTGTT, (SEQ ID NO: 13;Primer 2) CGTTAAAGGATCCTCGCTAAAAGCGATC, (SEQ ID NO: 14; (Primer 3)CTAGCCAAGCACTACGGACATTAGC, (SEQ ID NO: 15; (Primer 4)CGTAGTGCTTGGCTAGTAGATGCTGTT, (SEQ ID NO: 16; (Primer 5)CCTGGTGTTGGCAACCATGGTTG, (SEQ ID NO: 17; (Primer 6)GATCAACCATGGTTGCCAACACC, (SEQ ID N0: 18; (Primer 7)AAGGTGGACTCCAATCAGGGCACG, (SEQ ID NO: 19; (Primer 8)CGTGCCCTGATTGGAGTCCACCTT, (SEQ ID NO: 20; (Primer 9)GCGGTGTCATTTTCCACTTTTGG, (SEQ ID NO: 21; (Primer 10)CCAAAAGTGGAAAATGACACCGC, (SEQ ID NO: 22; (Primer 11)GGTATTCCAGAACAAGCCATTT, (SEQ ID NO: 23; (Primer 12)GCTTGTTCTGGAATACCAAGAGTG, (SEQ ID NO: 24; (Primer 13)ATAACCCCTATACCAGCCTAG, (SEQ ID NO: 25; (Primer 14)GCTGGTATTGGAGGGGTTATTACTATACCCCACAATTAAGTGGTTGGTCT TATCAGATG, (SEQ IDNO: 26; (Primer 15) CCATTCTACCCAATCCCAGCTGTA, and (SEQ ID NO: 27;(Primer 16) TACAGCTGGGATTGGGTAGAATGG.

Additionally provided in this invention are methods of diagnosinginfection by M. pneumoniae in a subject comprising contacting abiological sample from the subject with a polypeptide or antibody ofthis invention under conditions whereby an antigen/antibody complex canform; and detecting formation of an antigen/antibody complex, therebydiagnosing infection by M. pneumoniae in the subject.

Methods are also provided herein for diagnosing infection by M.pneumoniae in a subject comprising contacting a biological sample fromthe subject with a nucleic acid of this invention under conditionswhereby hybridization of nucleic acid molecules can occur; and detectinghybridization, thereby diagnosing infection by M. pneumoniae in thesubject.

Furthermore, the present invention provides methods of eliciting animmune response in a subject, comprising administering to the subject aneffective amount of a polypeptide and/or biologically active fragment ofa polypeptide of this invention and/or by administering to a subject aneffective amount of a nucleic acid comprising a nucleotide sequenceencoding a polypeptide and/or biologically active fragment of apolypeptide of this invention.

The present invention additionally provides methods of providing passiveimmunity to a subject, comprising administering to the subject aneffective amount of an antibody of this invention.

In further embodiments, the present invention provides methods oftreating and/or preventing infection by M. pneumoniae in a subject,comprising administering to the subject an effective amount of apolypeptide of this invention and/or an effective amount of abiologically active fragment of a polypeptide of this invention and/oran effective amount of a nucleic acid comprising a nucleotide sequenceencoding a polypeptide of this invention and/or an effective amount of anucleic acid comprising a nucleotide sequence encoding a biologicallyactive fragment of a polypeptide of this invention. Also provided aremethods of treating and/or preventing infection by M. pneumoniae in asubject, comprising administering to the subject an effective amount ofan antibody of this invention.

In yet further embodiments, the present invention provides methods ofidentifying substances having the ability to inhibit or enhance variousactivities of the polypeptides and/or biologically active fragments ofthis invention, including but not limited to, binding activity,translocating activity, immunogenic activity, ADP-ribosylating activityand/or cytopathology inducing activity. These methods are carried out bycontacting the polypeptides and/or biologically active fragments of thisinvention and/or the nucleic acids of this invention, with the substanceto be tested for inhibitory or enhancing activity, under conditionswhereby the inhibition or enhancement of activity can be detected, asdescribed herein.

Various other objectives and advantages of the present invention willbecome apparent from the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “a,” “an” or “the” can mean one or more than one. Forexample, “a” cell can mean a single cell or a multiplicity of cells.

The present invention is based on the discovery of polypeptides ofMycoplasma pneumoniae having the respective amino acid sequencedescribed herein and encoded by the nucleic acids described herein andthe identification of activities of these polypeptides and variousfragments or “domains” of these polypeptides. Characterization of thesepolypeptides and fragments indicates that the newly identified proteinis an exotoxin of Mycoplasma pneumoniae and it is referred to herein ascommunity acquired respiratory distress syndrome (CARDS) toxin. Thus,the present invention provides an isolated polypeptide comprising,consisting essentially of, and/or consisting of the amino acid sequenceof SEQ ID NO:2 (S1 isolate), an isolated polypeptide comprising,consisting essentially of, and/or consisting of the amino acid sequenceof SEQ ID NO:3 (JL isolate), an isolated polypeptide comprising,consisting essentially of, and/or consisting of the amino acid sequenceof SEQ ID NO:4 (RJL1 isolate), an isolated polypeptide comprising,consisting essentially of, and/or consisting of the amino acid sequenceof SEQ ID NO:5 (L2 isolate), an isolated polypeptide comprising,consisting essentially of, and/or consisting of the amino acid sequenceof SEQ ID NO:1 (reference sequence), and/or an isolated polypeptidecomprising, consisting essentially of, and/or consisting of the aminoacid sequence of SEQ ID NO:6 (composite sequence), either individuallyor in any combination.

The present invention further provides biologically active fragments ofthe polypeptides of this invention, as well as antibodies thatspecifically bind the polypeptides and/or fragments of the polypeptidesof this invention.

Further provided are nucleotide sequences that encode the polypeptidesand fragments of this invention. In particular, the present inventionprovides an isolated nucleic acid comprising, consisting essentially of,and/or consisting of the nucleotide sequence of SEQ ID NO:8 (S1isolate), an isolated nucleic acid comprising, consisting essentiallyof, and/or consisting of the nucleotide sequence of SEQ ID NO:10 (JLisolate), an isolated nucleic acid comprising, consisting essentially ofand/or consisting of the nucleotide sequence of SEQ ID NO:11 (RJL1isolate), an isolated nucleic acid comprising, consisting essentiallyof, and/or consisting of the nucleotides sequence of SEQ ID NO:9 (L2isolate), an isolated nucleic acid comprising, consisting essentiallyof, and/or consisting of the nucleotides sequence of SEQ ID NO:7(reference sequence), and/or an isolated nucleic acid comprising,consisting essentially of, and/or consisting of the nucleotide sequenceof SEQ ID NO:76 (composite sequence), either individually or in anycombination.

Additionally provided is a nucleic acid comprising, consistingessentially of, and/or consisting of a nucleotide sequence that encodesan amino acid sequence comprising, consisting essentially of, and/orconsisting of the amino acid sequence or a biologically active fragmentof the amino acid sequence of SEQ ID NO:2 (S1 isolate), a nucleic acidcomprising, consisting essentially of, and/or consisting of a nucleotidesequence that encodes an amino acid sequence comprising, consistingessentially of, and/or consisting of the amino acid sequence or abiologically active fragment of the amino acid sequence of SEQ ID NO:3(JL isolate), a nucleic acid comprising, consisting essentially ofand/or consisting of a nucleotide sequence that encodes an amino acidsequence comprising, consisting essentially of, and/or consisting of theamino acid sequence or a biologically active fragment of the amino acidsequence of SEQ ID NO:4 (RJL1 isolate), a nucleic acid comprising,consisting essentially of, and/or consisting of a nucleotides sequencethat encodes an amino acid comprising, consisting essentially of, and/orconsisting of the amino acid sequence or a biologically active fragmentof an amino acid sequence of SEQ ID NO:5 (L2 isolate), and/or a nucleicacid comprising, consisting essentially of, a nucleic acid comprising,consisting essentially of, and/or consisting of a nucleotides sequencethat encodes an amino acid comprising, consisting essentially of, and/orconsisting of the amino acid sequence or a biologically active fragmentof an amino acid sequence of SEQ ID NO:1 (reference sequence), and/or anucleic acid comprising, consisting essentially of, and/or consisting ofa nucleotide sequence encoding an amino acid sequence comprising,consisting essentially of, and/or consisting of the amino acid sequenceor a biologically active fragment of the amino acid sequence of SEQ IDNO:6 (composite sequence). Further provided herein is a nucleic acidthat is the complement of each and any of the nucleic acids of thisinvention.

Also provided herein are probes and primers for the detection of thenucleic acids of this invention, including

(SEQ ID NO: 12; Primer 1) TTTTTACATATGCCAAATCCTGTT, (SEQ ID NO: 13;Primer 2) CGTTAAAGGATCCTCGCTAAAAGCGATC, (SEQ ID NO: 14; (Primer 3)CTAGCCAAGCACTACGGACATTAGC, (SEQ ID NO: 15; (Primer 4)CGTAGTGCTTGGCTAGTAGATGCTGTT, (SEQ ID NO: 16; (Primer 5)CCTGGTGTTGGCAACCATGGTTG, (SEQ ID NO: 17; (Primer 6)GATCAACCATGGTTGCCAACACC, (SEQ ID NO: 18; (Primer 7)AAGGTGGACTCCAATCAGGGCACG, (SEQ ID NO: 19; (Primer 8)CGTGCCCTGATTGGAGTCCACCTT, (SEQ ID NO: 20; (Primer 9)GCGGTGTCATTTTCCACTTTTGG, (SEQ ID NO: 21; (Primer 10)CCAAAAGTGGAAAATGACACCGC, (SEQ ID NO: 22; (Primer 11)GGTATTCCAGAACAAGCCATTT, (SEQ ID NO: 23; (Primer 12)GCTTGTTCTGGAATACCAAGAGTG, (SEQ ID NO: 24; (Primer 13)ATAACCCCTATACCAGCCTAG, (SEQ ID NO:25; (Primer 14)GCTGGTATTGGAGGGGTTATTACTATACCCCACAATTAAGTGGTTGGTCT TATCAGATG, (SEQ IDNO: 26; (Primer 15) CCATTCTACCCAATCCCAGCTGTA, and (SEQ ID NO: 27;(Primer 16) TACAGCTGGGATTGGGTAGAATGG,alone and/or in any combination. The present invention further providesas additional embodiments without limitation, other oligonucleotideslisted in this application and in the Sequence Listing attached hereto.

“Isolated” as used herein means the nucleic acid or polypeptide of thisinvention is sufficiently free of contaminants or cell components withwhich nucleic acids or polypeptides normally occur. “Isolated” does notmean that the preparation is technically pure (homogeneous), but it issufficiently pure to provide the nucleic acid or polypeptide in a formin which it can be used therapeutically.

“Epitope” or “antigenic epitope” or “antigenic peptide” as used hereinmeans a specific amino acid sequence of limited length which, whenpresent in the proper conformation, provides a reactive site for anantibody or T cell receptor. The identification of epitopes on antigenscan be carried out by immunology protocols that are well known in theart.

As used herein, the term “polypeptide” or “protein” is used to describea chain of amino acids that correspond to those encoded by a nucleicacid. A polypeptide of this invention can be a peptide, which usuallydescribes a chain of amino acids of from two to about 30 amino acids.The term polypeptide as used herein also describes a chain of aminoacids having more than 30 amino acids and can be a fragment or domain ofa protein or a full length protein. Furthermore, as used herein, theterm polypeptide can refer to a linear chain of amino acids or it canrefer to a chain of amino acids that has been processed and folded intoa functional protein. It is understood, however, that 30 is an arbitrarynumber with regard to distinguishing peptides and polypeptides and theterms can be used interchangeably for a chain of amino acids. Thepolypeptides of the present invention are obtained by isolation andpurification of the polypeptides from cells where they are producednaturally, by enzymatic (e.g., proteolytic) cleavage, and/orrecombinantly by expression of nucleic acid encoding the polypeptides orfragments of this invention. The polypeptides and/or fragments of thisinvention can also be obtained by chemical synthesis or other knownprotocols for producing polypeptides and fragments.

The amino acid sequences disclosed herein are presented in the amino tocarboxy direction, from left to right. Nucleotide sequences arepresented herein by single strand only, in the 5′ to 3′ direction, fromleft to right. However, it is intended that the nucleic acids of thisinvention can be either single or double stranded (i.e., including thecomplementary nucleic acid). A nucleic acid of this invention can be thecomplement of a nucleic acid described herein.

A “biologically active fragment” includes a polypeptide of thisinvention that comprises a sufficient number of amino acids to have oneor more of the biological activities of the polypeptides of thisinvention. Such biological activities can include, but are not limitedto, in any combination, binding activity, translocating activity,immunogenic activity, ADP-ribosylating activity, and/or cytopathologyinducing activity, as well as any other activity now known or lateridentified for the polypeptides and/or fragments of this invention. Afragment of a polypeptide of this invention can be produced by methodswell known and routine in the art. Fragments of this invention can beproduced, for example, by enzymatic or other cleavage of naturallyoccurring peptides or polypeptides or by synthetic protocols that arewell known. Such fragments can be tested for one or more of thebiological activities of this invention according to the methodsdescribed herein, which are routine methods for testing activities ofpolypeptides, and/or according to any art-known and routine methods foridentifying such activities. Such production and testing to identifybiologically active fragments of the polypeptides described herein wouldbe well within the scope of one of ordinary skill in the art and wouldbe routine.

Fragments of the polypeptides of this invention are preferably at leastabout ten amino acids in length and retain one or more of the biologicalactivities and/or the immunological activities of the CARDS toxin.Examples of the fragments of this invention include, but are notintended to be limited to, the following fragments identified by theamino acid number as shown in the Sequence Listing for each of theisolates of SEQ ID NO:2 (S1 isolate), SEQ ID NO:3 (JL isolate), SEQ IDNO:4 (RJL1 isolate), SEQ ID NO:5 (L2 isolate), SEQ ID NO:6 (compositesequence) and SEQ ID NO:1 (reference sequence): Amino acids 1-10, 10-20,20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 110-120,120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200,200-210, 210-220, 220-230, 230-240, 240-250, 250-260, 260-270, 270-280,280-290, 290-300, 300-310, 310-320, 320-330, 330-340, 340-350, 350-360,360-370, 370-380, 380-390, 390-400, 400-410, 410-420, 420-430, 430-440,440-450, 450-460, 460-470, 470-480, 480-490, 490-500, 500-510, 510-520,520-530, 530-540, 540-550, 550-560, 560-570, 570-580, 580-591, 1-25,1-50, 1-67, 1-75, 1-100, 1-125, 1-135, 1-145, 1-150, 1-160, 1-170,1-180, 1-190, 1-200, 1-250, 1-300, 1-350, 1-400, 1-450, 1-500, 68-180,183-123, 500-591, 450-591, 400-591, 350-591, 300-591, 250-591, 200-591,150-591, 100-591, 50-591, 50-100, 100-200, 200-300, 300-400, 400-500,500-591, 550-591.

It is understood that this list is exemplary only and that a fragment ofthis invention can be any amino acid sequence containing any combinationof contiguous amino acids that are numbered in the Sequence Listing asamino acids 1 through 591 even if that combination is not specificallyrecited as an example herein. It is also understood that these fragmentscan be combined in any order or amount. For example, fragment 1-10 canbe combined with fragment 10-20 to produce a fragment of amino acids1-20. Also fragments can be present in multiple numbers and in anycombination in a fragment of this invention. Thus, for example, fragment1-150 can be combined with a second fragment 1-150 and/or combined withfragment 400-500 to produce a fragment of this invention. Otherexemplary fragments of this invention include the domains of the CARDStoxin described herein [e.g., domain 1 (N terminal 249 amino acids),domain 2 (256 amino acids) and domain 3 (247 amino acids at carboxyterminus)].

The term “homology” as used herein refers to a degree of similaritybetween two or more sequences. There may be partial homology or completehomology (i.e., identity). A partially complementary sequence that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid is referred to as “substantially homologous.” Theinhibition of hybridization of the completely complementary sequence tothe target sequence can be examined using a hybridization assay(Southern or Northern blot, solution hybridization and the like) underconditions of low stringency. A substantially homologous sequence orhybridization probe will compete for and inhibit the binding of acompletely homologous sequence to the target sequence under conditionsof low stringency, as this term is known in the art. This is not to saythat conditions of low stringency are such that non-specific binding ispermitted; low stringency conditions require that the binding of twosequences to one another be a specific (i.e., selective) interaction.The absence of non-specific binding can be tested by the use of a secondtarget sequence that lacks even a partial degree of complementarity(e.g., less than about 30% identity). In the absence of non-specificbinding, the probe will not hybridize to the second non-complementarytarget sequence.

The term “hybridization” as used herein refers to any process by which afirst strand of nucleic acid binds with a second strand of nucleic acidthrough base pairing. Nucleic acids encoding the polypeptides and/orfragments of this invention can be detected by DNA-DNA or DNA-RNAhybridization or amplification using probes, primers and/or fragments ofpolynucleotides encoding the polypeptides and/or fragments of thisinvention and/or designed to detect and/or amplify the nucleic acids ofthis invention.

The term “hybridization complex” as used herein refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells and/or nucleic acids have beenfixed).

The term “nucleotide sequence” refers to a heteropolymer of nucleotidesor the sequence of these nucleotides. The terms “nucleic acid,”“oligonucleotide” and “polynucleotide” are also used interchangeablyherein to refer to a heteropolymer of nucleotides. Generally, nucleicacid segments provided by this invention may be assembled from fragmentsof the genome and short oligonucleotide linkers, or from a series ofoligonucleotides, or from individual nucleotides, to provide a syntheticnucleic acid which is capable of being expressed in a recombinanttranscriptional unit comprising regulatory elements derived from amicrobial or viral operon, or a eukaryotic gene. Nucleic acids of thisinvention can comprise a nucleotide sequence that can be identical insequence to the sequence which is naturally occurring or, due to thewell-characterized degeneracy of the nucleic acid code, can includealternative codons which encode the same amino acid as that which isfound in the naturally occurring sequence. Furthermore, nucleic acids ofthis invention can comprise nucleotide sequences that can include codonswhich represent conservative substitutions of amino acids as are wellknown in the art, such that the biological activity of the resultingpolypeptide and/or fragment is retained.

The term “probe” or “primer” includes naturally occurring or recombinantor chemically synthesized single- and/or double-stranded nucleic acids.They can be labeled for detection by nick translation, Klenow fill-inreaction, PCR or other methods well known in the art. Probes and primersof the present invention, their preparation and/or labeling aredescribed in Sambrook et al. 1989. Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, NY and Ausubel et al. 1989.Current Protocols in Molecular Biology, John Wiley & Sons, New YorkN.Y., both of which are incorporated herein by reference in theirentirety for these teachings.

The term “stringent” as used here refers to hybridization conditionsthat are commonly understood in the art to define the conditions of thehybridization procedure. Stringency conditions can be low, high ormedium, as those terms are commonly know in the art and well recognizedby one of ordinary skill. In various embodiments, stringent conditionscan include, for example, highly stringent (i.e., high stringency)conditions (e.g., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7%sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in0.1×SSC/0.1% SDS at 68° C.), and/or moderately stringent (i.e., mediumstringency) conditions (e.g., washing in 0.2×SSC/0.1% SDS at 42° C.).

“Amplification” as used herein includes the production of multiplecopies of a nucleic acid molecule and is generally carried out usingpolymerase chain reaction (PCR) and/or other amplification technologiesas are well known in the art (Dieffenbach and Dveksler. 1995. PCRPrimer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

As used herein, the term “antibody” includes intact immunoglobinmolecules as well as fragments thereof, such as Fab, F(ab′)2, and Fc,which are capable of binding the epitopic determinant of an antigen(i.e., antigenic determinant). Antibodies that bind the polypeptides ofthis invention are prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or fragment used to immunize an animal can be derived fromenzymatic cleavage, recombinant expression, isolation from biologicalmaterials, synthesis, etc., and can be conjugated to a carrier protein,if desired. Commonly used carriers that are chemically coupled topeptides and proteins for the production of antibody include, but arenot limited to, bovine serum albumin, thyroglobulin and keyhole limpethemocyanin. The coupled peptide or protein is then used to immunize theanimal (e.g., a mouse, rat, or rabbit). The polypeptide or peptideantigens can also be administered with an adjuvant, as described hereinand as otherwise known in the art.

The term “antibody” or “antibodies” as used herein refers to all typesof immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibodycan be monoclonal or polyclonal and can be of any species of origin,including, for example, mouse, rat, rabbit, horse, goat, sheep or human,or can be a chimeric or humanized antibody. See, e.g., Walker et al.,Molec. Immunol. 26:403-11 (1989). The antibodies can be recombinantmonoclonal antibodies produced according to the methods disclosed inU.S. Pat. No. 4,474,893 or U.S. Pat. No. 4,816,567. The antibodies canalso be chemically constructed according to the method disclosed in U.S.Pat. No. 4,676,980. The antibody can further be a single chain antibodyor bispecific antibody.

Antibody fragments included within the scope of the present inventioninclude, for example, Fab, F(ab′)2, and Fc fragments, and thecorresponding fragments obtained from antibodies other than IgG. Suchfragments can be produced by known techniques.

For example, F(ab′)2 fragments can be produced by pepsin digestion ofthe antibody molecule, and Fab fragments can be generated by reducingthe disulfide bridges of the F(ab′)2 fragments. Alternatively, Fabexpression libraries can be constructed to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity(Huse et al., (1989) Science 254:1275-1281).

Monoclonal antibodies can be produced in a hybridoma cell line accordingto the technique of Kohler and Milstein, (1975) Nature 265:495-97. Forexample, a solution containing the appropriate antigen can be injectedinto a mouse and, after a sufficient time, the mouse sacrificed andspleen cells obtained. The spleen cells are then immortalized by fusingthem with myeloma cells or with lymphoma cells, typically in thepresence of polyethylene glycol, to produce hybridoma cells. Thehybridoma cells are then grown in a suitable medium and the supernatantscreened for monoclonal antibodies having the desired specificity.Monoclonal Fab fragments can be produced in bacterial cell such as E.coli by recombinant techniques known to those skilled in the art. See,e.g., W. Huse, (1989) Science 246:1275-81.

Antibodies can also be obtained by phage display techniques known in theart or by immunizing a heterologous host with a cell containing anepitope of interest.

The term “sample” as used herein is used in its broadest sense. Abiological sample suspected of containing a polypeptide, fragment,antibody and/or nucleic acid of this invention can be any biologicalfluid, an extract from a cell, an extracellular matrix isolated from acell, a cell (in solution or bound to a solid support), a tissue, atissue print, and the like.

“Effective amount” refers to an amount of a compound or composition ofthis invention that is sufficient to produce a desired effect, which canbe a therapeutic effect.

The effective amount will vary with the age, general condition of thesubject, the severity of the condition being treated, the particularagent administered, the duration of the treatment, the nature of anyconcurrent treatment, the pharmaceutically acceptable carrier used, andlike factors within the knowledge and expertise of those skilled in theart. As appropriate, an “effective amount” in any individual case can bedetermined by one of ordinary skill in the art by reference to thepertinent texts and literature and/or by using routine experimentation.(See, for example, Remington, The Science And Practice of Pharmacy (20thed. 2000)).

A “pharmaceutically acceptable” component such as a salt, carrier,excipient or diluent of a composition according to the present inventionis a component that (i) is compatible with the other ingredients of thecomposition in that it can be combined with the compositions of thepresent invention without rendering the composition unsuitable for itsintended purpose, and (ii) is suitable for use with subjects as providedherein without undue adverse side effects (such as toxicity, irritation,and allergic response). Side effects are “undue” when their riskoutweighs the benefit provided by the composition. Non-limiting examplesof pharmaceutically acceptable components include, without limitation,any of the standard pharmaceutical carriers such as phosphate bufferedsaline solutions, water, emulsions such as oil/water emulsion,microemulsions and various types of wetting agents.

“Treat,” “treating” or “treatment” refers to any type of action thatimparts a modulating effect, which, for example, can be a beneficialeffect, to a subject afflicted with a disorder, disease or illness,including improvement in the condition of the subject (e.g., in one ormore symptoms), delay in the progression of the condition, prevention ordelay of the onset of the disorder, and/or change in clinicalparameters, disease or illness, etc., as would be well known in the art.

A subject of this invention includes any animal susceptible to infectionby Mycoplasma pneumoniae. Such a subject can be a mammal and inparticular embodiments, is a human. A “subject in need thereof” is asubject known to be, or suspected of being, infected with Mycoplasmapneumoniae. A subject of this invention can also include a subject notpreviously known or suspected to be infected by Mycoplasma pneumoniae orin need of treatment for Mycoplasma pneumoniae infection. For example, asubject of this invention can be administered the compositions of thisinvention even if it is not known or suspected that the subject isinfected with Mycoplasma pneumoniae (e.g., prophylactically). A subjectof this invention is also a subject known or believed to be at risk ofinfection by Mycoplasma pneumoniae.

In certain embodiments, the fragments and/or polypeptides of thisinvention can be fused with a “carrier” protein or peptide to produce afusion protein. For example, the carrier protein or peptide can be fusedto a polypeptide and/or fragment of this invention to increase thestability thereof (e.g., decrease the turnover rate) in the cell and/orsubject. Exemplary carrier proteins include, but are not limited to,glutathione-S-transferase or maltose-binding protein. The carrierprotein or peptide can alternatively be a reporter protein. For example,the fusion protein can comprise a polypeptide and/or fragment of thisinvention and a reporter protein or peptide (e.g., Green FluorescentProtein, β-glucoronidase, β-galactosidase, luciferase, and the like) foreasy detection of transformed cells and transgene expression. As afurther alternative, the fusion protein attached to the polypeptidesand/or fragments and a carrier protein or peptide can be targeted to asubcellular compartment of interest, i.e., to affect the co-localizationof the polypeptide and/or fragment. Any suitable carrier protein as iswell known in the art can be used to produce a fusion protein of thisinvention.

The polypeptides and/or fragments of the present invention can 1) beused in assays to determine the biological activity of other proteins orpeptides; 2) be included in a panel of multiple proteins forhigh-throughput screening; 3) be used to raise antibodies or to elicitan immune response; 4) be used as a reagent (including the labeledreagent) in assays designed to quantitatively determine levels of theprotein (or its binding partner or receptor) in biological fluids; and5) be used as markers for tissues in which the corresponding protein ispreferentially expressed (either constitutively or at a particular stageof tissue differentiation or development or in a disease state). Any orall of these research utilities are capable of being developed intoreagent grade or kit format for commercialization as research products.Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include MolecularCloning: A Laboratory Manual, 2d ed., Cold Spring Harbor LaboratoryPress, Sambrook et al., eds. (1989) and Methods in Enzymology: Guide toMolecular Cloning Techniques, Academic Press, Berger and Kimmel eds.(1987).

A variety of protocols for detecting the presence of and/or measuringthe amount of polypeptides, fragments and/or peptides in a sample, usingeither polyclonal or monoclonal antibodies specific for the polypeptide,fragment and/or peptide are known in the art. Examples of such protocolsinclude, but are not limited to, enzyme immunoassays (EIA),agglutination assays, immunoblots (Western blot; dot/slot blot, etc.),radioimmunoassays (RIA), immunodiffusion assays, chemiluminescenceassays, antibody library screens, expression arrays, enzyme-linkedimmunosorbent assays (ELISA), radioimmunoassays (RIA),immunoprecipitation, Western blotting, competitive binding assays,immunofluorescence, immunohistochemical stainingprecipitation/flocculation assays and fluorescence-activated cellsorting (FACS). These and other assays are described, among otherplaces, in Hampton et al. (Serological Methods, a Laboratory Manual, APSPress, St Paul, Minn. (1990)) and Maddox et al. (J. Exp. Med.158:1211-1216 (1993)).

Furthermore, a number of assays for detection and/or amplification ofnucleic acid sequences are well known in the art. Additionally, a widevariety of labeling and conjugation techniques are known in the art thatare used in various nucleic acid detection and amplification assays.Methods for producing labeled hybridization probes and/or PCR or otherligation primers for detecting and/or amplifying nucleic acid sequencescan include, for example, oligolabeling, nick translation andend-labeling, as well as other well known methods. Alternatively,nucleic acid sequences encoding the polypeptides of this invention,and/or any functional fragment thereof, can be cloned into a plasmid orvector for detection and amplification. Such plasmids and vectors arewell known in the art and are commercially available. It is alsocontemplated that the methods of this invention can be conducted using avariety of commercially-available kits (e.g., Pharmacia & Upjohn;Promega; U.S. Biochemical Corp.). Suitable reporter molecules or labels,which can be used for ease of detection, include, for example,radionuclides, enzymes, fluorescence agents, chemiluminescence agentsand chromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles and the like as are well known in the art.

The present invention further includes isolated polypeptides, peptides,proteins, fragments, domains and/or nucleic acid molecules that aresubstantially equivalent to those described for this invention. As usedherein, “substantially equivalent” can refer both to nucleic acid andamino acid sequences, for example a mutant sequence, that varies from areference sequence by one or more substitutions, deletions, oradditions, the net effect of which does not result in an undesirableadverse functional dissimilarity between reference and subjectsequences. In some embodiments, this invention can include substantiallyequivalent sequences that have an adverse functional dissimilarity. Forpurposes of the present invention, sequences having equivalentbiological activity and equivalent expression characteristics areconsidered substantially equivalent.

The invention further provides homologs, as well as methods of obtaininghomologs, of the polypeptides and/or fragments of this invention fromother strains of Mycoplasma and/or other organisms. As used herein, anamino acid sequence or protein is defined as a homolog of a polypeptideor fragment of the present invention if it shares significant homologyto one of the polypeptides and/or fragments of the present invention.Significant homology means at least 75%, 80%, 85%, 90%, 95%, 98% and/or100% homology with another amino acid sequence. Specifically, by usingthe nucleic acids disclosed herein as a probe or as primers, andtechniques such as PCR amplification and colony/plaque hybridization,one skilled in the art can identify homologs of the polypeptides and/orfragments of this invention in Mycoplasma and/or other organisms.

The present invention also provides an antibody that specifically bindsthe polypeptides and/or biologically active fragments of this invention,as well as a method of making an antibody specific for a polypeptideand/or fragment of this invention comprising: a) immunizing an animalwith a polypeptide and/or fragment of this invention under conditionswhereby the animal produces antibodies that specifically bind thepolypeptide and/or fragment of this invention; and b) removingbiological materials comprising the antibodies from the animal. Alsoprovided herein is an antibody produced by the methods set forth herein.

Antibodies of this invention can be generated using methods that arewell known in the art. Such antibodies and immunoglobulin molecules ofthis invention can include, but are not limited to, polyclonalantibodies, monoclonal antibodies, chimeric antibodies, humanizedantibodies, single chain antibodies (e.g., scFv), Fab fragments, andfragments produced by a Fab expression library.

In general, techniques for preparing polyclonal and monoclonalantibodies as well as hybridomas capable of producing a desired antibodyare well known in the art. Any animal known to produce antibodies can beimmunized with a polypeptide, fragment and/or antigenic epitope of thisinvention. Methods for immunization of animals to produce antibodies arewell known in the art. For example, such methods can includesubcutaneous or interperitoneal injection of the polypeptide, fragmentand/or antigenic epitope of this invention.

The polypeptide, fragment or antigenic epitope that is used as animmunogen can be modified or administered in an adjuvant in order toincrease antigenicity. Methods of increasing the antigenicity of aprotein or peptide are well known in the art and include, but are notlimited to, coupling the antigen with a heterologous protein (such asglobulin or β-galactosidase) or through the inclusion of an adjuvantduring immunization.

For example, for the production of antibodies, various hosts includinggoats, rabbits, rats, mice, humans, and others, can be immunized byinjection with the polypeptides and/or fragments of this invention, withor without a carrier protein. Additionally, various adjuvants may beused to increase the immunological response. Such adjuvants include, butare not limited to, Freund's complete and incomplete adjuvants, mineralgels such as aluminum hydroxide, and surface-active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, and dinitrophenol. Among adjuvants used inhumans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum areespecially preferable.

Polypeptides, peptides and/or fragments of this invention used asantigens to produce the antibodies of this invention can have an aminoacid sequence consisting of at least five amino acids and in certainembodiments, at least ten amino acids. In one embodiment, the antigen isidentical to a portion of the amino acid sequence of the naturalprotein, and it can contain the entire amino acid sequence of a small,naturally-occurring molecule. Short stretches of the polypeptides and/orfragments of this invention can be fused with all or a fragment ofanother protein that acts as a carrier protein (e.g., keyhole limpethemocyanin) and antibodies can be produced against the chimericpolypeptide or peptide.

Monoclonal antibodies to the polypeptides and/or fragments of thisinvention are prepared using any technique, which provides for theproduction of antibody molecules by continuous cell lines in culture.These include, but are not limited to, the hybridoma technique, thehuman B-cell hybridoma technique, and the EBV-hybridoma technique(Kohler et al. 1975. Nature 256:495-497; Kozbor et al. 1985. J. Immunol.Methods 81:31-42; Cote et al. 1983. Proc. Natl. Acad. Sci. 80:2026-2030;Cole et al. 1984. Mol. Cell Biol. 62:109-120).

For example, to produce monoclonal antibodies, spleen cells from theimmunized animal are removed, fused with myeloma cells, and cultured inselective medium to become monoclonal antibody-producing hybridomacells, according to techniques routine in the art. Any one of a numberof methods well known in the art can be used to identify the hybridomacell, which produces an antibody with the desired characteristics. Theseinclude screening the hybridomas by ELISA assay, Western blot analysis,or radioimmunoassay. Hybridomas secreting the desired antibodies arecloned and the class and subclass are identified using standardprocedures known in the art.

For polyclonal antibodies, antibody-containing serum is isolated fromthe immunized animal and is screened for the presence of antibodies withthe desired specificity using any of the well known procedures asdescribed herein.

The present invention further provides antibodies of this invention indetectably labeled form. Antibodies can be detectably labeled throughthe use of radioisotopes, affinity labels (such as biotin, avidin,etc.), enzymatic labels (such as horseradish peroxidase, alkalinephosphatase, etc.) fluorescence labels (such as FITC or rhodamine,etc.), paramagnetic atoms, gold beads, etc. Such labeling procedures arewell-known in the art. The labeled antibodies of the present inventioncan be used for in vitro, in vivo, and in situ assays to identify apolypeptide and/or fragment of this invention in a sample.

In some embodiments, the present invention further provides theabove-described antibodies immobilized on a solid support (e.g., beads,plates, slides or wells formed from materials such as latex orpolystyrene). Examples of such solid supports include plastics such aspolycarbonate, complex carbohydrates such as agarose and sepharose,acrylic resins and such as polyacrylamide and latex beads. Techniquesfor coupling antibodies to such solid supports are well known in the art(Weir et al., Handbook of Experimental Immunology 4th Ed., BlackwellScientific Publications, Oxford, England, Chapter 10 (1986)). Antibodiescan likewise be conjugated to detectable groups such as radiolabels(e.g., ³⁵S, ¹²⁵I, ¹³¹I) enzyme labels (e.g., horseradish peroxidase,alkaline phosphatase), and fluorescence labels (e.g., fluorescein) inaccordance with known techniques. Determination of the formation of anantibody/antigen complex in the methods of this invention can be bydetection of, for example, precipitation, agglutination, flocculation,radioactivity, color development or change, fluorescence, luminescence,etc., as is well know in the art.

In addition, techniques developed for the production of chimericantibodies or humanized antibodies by splicing mouse antibody genes tohuman antibody genes to obtain a molecule with appropriate antigenspecificity and biological activity can be used (Morrison et al. 1984.Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger et al. 1984. Nature312:604-608; Takeda et al. 1985. Nature 314:452-454). Alternatively,techniques described for the production of single chain antibodies canbe adapted, using methods known in the art, to produce single chainantibodies specific for the polypeptides and fragments of thisinvention. Antibodies with related specificity, but of distinctidiotypic composition, can be generated by chain shuffling from randomcombinatorial immunoglobin libraries (Burton 1991. Proc. Natl. Acad.Sci. 88:11120-3).

Antibody fragments that specifically bind the polypeptides and/orfragments of this invention can also be generated. For example, suchfragments include, but are not limited to, the F(ab)₂ fragments that canbe produced by pepsin digestion of the antibody molecule and the Fabfragments that can be generated by reducing the disulfide bridges of theF(ab)₂ fragments. Alternatively, Fab expression libraries may beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity (Huse et al. 1989. Science254:1275-1281).

Various immunoassays can be used for screening to identify antibodieshaving the desired specificity for the proteins and peptides of thisinvention. Numerous protocols for competitive binding orimmunoradiometric assays using either polyclonal or monoclonalantibodies with established specificity are well known in the art. Suchimmunoassays typically involve the measurement of complex formationbetween an antigen and its specific antibody (e.g., antigen/antibodycomplex formation). For example, a two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on the proteins or peptides of this inventioncan be used, as well as a competitive binding assay.

It is further contemplated that the present invention provides kits fordetection of the polypeptides and/or fragments of this invention in asample. In one embodiment, the kit can comprise one or more antibodiesof this invention, along with suitable buffers, wash solutions and/orother reagents for the detection of antibody/antigen complex formation.In an alternative embodiment, a kit of this invention can comprise apolypeptide, an antigenic peptide of the polypeptide of this invention,a fragment of this invention and/or an antigenic peptide of a fragmentof this invention, along with suitable buffers, wash solutions and/orother reagents for the detection of antibody/antigen complex formation.

The present invention further provides a kit for the detection ofnucleic acid encoding the polypeptides and/or fragments of thisinvention. For example, in one embodiment, the kit can comprise one ormore nucleic acids of this invention, along with suitable buffers, washsolutions and/or other reagents for the detection of hybridizationcomplex formation.

It would be well understood by one of ordinary skill in the art that thekits of this invention can comprise one or more containers and/orreceptacles to hold the reagents (e.g., antibodies, antigens, nucleicacids) of the kit, along with appropriate buffers and/or wash solutionsand directions for using the kit, as would be well known in the art.Such kits can further comprise adjuvants and/or other immunostimulatoryor immunomodulating agents, as are well known in the art.

In further embodiments, the nucleic acids encoding the polypeptidesand/or fragments of this invention can be part of a recombinant nucleicacid construct comprising any combination of restriction sites and/orfunctional elements as are well known in the art which facilitatemolecular cloning and other recombinant DNA manipulations. Thus, thepresent invention further provides a recombinant nucleic acid constructcomprising a nucleic acid encoding a polypeptide and/or biologicallyactive fragment of this invention.

The present invention further provides a vector comprising a nucleicacid encoding a polypeptide and/or fragment of this invention. Thevector can be an expression vector which contains all of the geneticcomponents required for expression of the nucleic acid in cells intowhich the vector has been introduced, as are well known in the art. Theexpression vector can be a commercial expression vector or it can beconstructed in the laboratory according to standard molecular biologyprotocols. The expression vector can comprise viral nucleic acidincluding, but not limited to, vaccinia virus, adenovirus, retrovirusand/or adeno-associated virus nucleic acid. The nucleic acid or vectorof this invention can also be in a liposome or a delivery vehicle, whichcan be taken up by a cell via receptor-mediated or other type ofendocytosis.

The nucleic acid of this invention can be in a cell, which can be a cellexpressing the nucleic acid whereby a polypeptide and/or biologicallyactive fragment of this invention is produced in the cell. In addition,the vector of this invention can be in a cell, which can be a cellexpressing the nucleic acid of the vector whereby a polypeptide and/orbiologically active fragment of this invention is produced in the cell.

It is also contemplated that the nucleic acids and/or vectors of thisinvention can be present in a host animal (e.g., a transgenic animal),which expresses the nucleic acids of this invention and produces thepolypeptides and/or fragments of this invention.

The nucleic acid encoding the polypeptide and/or fragment of thisinvention can be any nucleic acid that functionally encodes thepolypeptides and/or fragments of this invention. To functionally encodethe polypeptides and/or fragments (i.e., allow the nucleic acids to beexpressed), the nucleic acid of this invention can include, for example,expression control sequences, such as an origin of replication, apromoter, an enhancer and necessary information processing sites, suchas ribosome binding sites, RNA splice sites, polyadenylation sites andtranscriptional terminator sequences.

Preferred expression control sequences are promoters derived frommetallothionine genes, actin genes, immunoglobulin genes, CMV, SV40,adenovirus, bovine papilloma virus, etc. A nucleic acid encoding aselected polypeptide and/or fragment can readily be determined basedupon the genetic code for the amino acid sequence of the selectedpolypeptide and/or fragment and many nucleic acids will encode anyselected polypeptide and/or fragment. Modifications in the nucleic acidsequence encoding the polypeptide and/or fragment are also contemplated.Modifications that can be useful are modifications to the sequencescontrolling expression of the polypeptide and/or fragment to makeproduction of the polypeptide and/or fragment inducible or repressibleas controlled by the appropriate inducer or repressor. Such methods arestandard in the art. The nucleic acid of this invention can be generatedby means standard in the art, such as by recombinant nucleic acidtechniques and by synthetic nucleic acid synthesis or in vitro enzymaticsynthesis.

In yet further embodiments, the present invention provides a D1 domainof CARDS Toxin comprising, consisting essentially of and/or consistingof the amino acid sequence of SEQ ID NO:69 and/or SEQ ID NO:75, a D2domain of CARDS Toxin comprising, consisting essentially of, orconsisting of the amino acid sequence of SEQ ID NO: 70, and/or a D3domain of CARDS Toxin comprising, consisting essentially of, and/orconsisting of the amino acid sequence of SEQ ID NO:71, in anycombination.

Further provided herein is an isolated nucleic acid encoding the aminoacid sequence of the domains D1, D2 and D3 of this invention. As oneexample, a nucleic acid encoding the domain D1 can comprise, consist ofand/or consist essentially of the nucleotide sequence of SEQ ID NO:74.

Additionally provided herein are antibodies that specifically binddomain D1, D2 and/or D3 of the CARDS Toxin of this invention. The domainpeptides can be used as antigens for the production of antibodies, whichcan be polyclonal and/or monoclonal, according to well known protocols.The domain peptides and antibodies can be used in the methods describedherein for the detection of M. pneumoniae antibodies and proteins and/orfor diagnosis of M. pneumoniae infection, as well as in therapeuticmethods to treat M. pneumoniae infection and related diseases asdescribed herein.

The present invention further provides a method of producing apolypeptide and/or biologically active fragment according to the methodsset forth in the Examples provided herein, and as are well known in theart for polypeptide synthesis. In one embodiment, a nucleic acidencoding the polypeptides and/or fragments of this invention can besynthesized according to standard nucleic acid synthesis protocols andthe nucleic acid can be expressed according to methods well known forexpression of nucleic acid. The resulting polypeptide and/or fragmentcan then be removed from the expression system by standard isolation andpurification procedures and tested for any of the various biologicalactivities described herein according to methods as taught herein aswell as methods routine in the art.

The present invention also provides a method for producing thepolypeptides and/or biologically active fragments of this inventioncomprising producing the cells of this invention which contain thenucleic acids or vectors of this invention as exogenous nucleic acid;culturing the cells under conditions whereby the exogenous nucleic acidin the cell can be expressed and the encoded polypeptide and/or fragmentcan be produced; and isolating the polypeptide and/or fragment from thecell. Thus, it is contemplated that the polypeptides and/or fragments ofthis invention can be produced in quantity in vitro in eitherprokaryotic or eukaryotic expression systems as are well known in theart.

As one example, for expression in a prokaryotic system, there arenumerous E. coli (Escherichia coli) expression vectors known to one ofordinary skill in the art useful for the expression of nucleic acid thatencodes polypeptides. Other microbial hosts suitable for use includebacilli, such as Bacillus subtilis, and other enterobacteria, such asSalmonella, Serratia, as well as various Pseudomonas species. Theseprokaryotic hosts can support expression vectors that will typicallycontain expression control sequences compatible with the host cell(e.g., an origin of replication). In addition, any number of a varietyof well-known promoters can be present, such as the lactose promotersystem, a tryptophan (Trp) promoter system, a beta-lactamase promotersystem, or a promoter system from phage lambda. The promoters willtypically control expression, optionally with an operator sequence andhave ribosome binding site sequences for example, for initiating andcompleting transcription and translation. If necessary, an aminoterminal methionine can be provided by insertion of a Met codon 5′ andin-frame with the polypeptide. Also, the carboxy-terminal extension ofthe polypeptide can be removed using standard oligonucleotidemutagenesis procedures.

The nucleic acid sequences can be expressed in hosts after the sequenceshave been positioned to ensure the functioning of an expression controlsequence. These expression vectors are typically replicable in the hostorganisms either as episomes or as an integral part of the hostchromosomal DNA. Commonly, expression vectors can contain selectionmarkers, e.g., tetracycline resistance or hygromycin resistance, topermit detection and/or selection of those cells transformed with thedesired nucleic acid sequences.

As another example, for eukaryotic system expression, a yeast expressionsystem can be used. There are several advantages to yeast expressionsystems. First, evidence exists that polypeptides produced in a yeastexpression system exhibit correct disulfide pairing. Second,post-translational glycosylation is efficiently carried out by yeastexpression systems. The Saccharomyces cerevisiae pre-pro-alpha-factorleader region (encoded by the MFα-1 gene) is routinely used to directprotein secretion from yeast. The leader region of pre-pro-alpha-factorcontains a signal peptide and a pro-segment, which includes arecognition sequence for a yeast protease encoded by the KEX2 gene. Thisenzyme cleaves the precursor protein on the carboxyl side of a Lys-Argdipeptide cleavage-signal sequence. The polypeptide coding sequence canbe fused in-frame to the pre-pro-alpha-factor leader region. Thisconstruct is then put under the control of a strong transcriptionpromoter, such as the alcohol dehydrogenase I promoter or a glycolyticpromoter. The coding sequence is followed by a translation terminationcodon, which is followed by transcription termination signals.Alternatively, the coding sequence of interest can be fused to a secondpolypeptide coding sequence, such as Sj26 or β-galactosidase, used tofacilitate purification of the resulting fusion polypeptide by affinitychromatography. The insertion of protease cleavage sites to separate thecomponents of the fusion polypeptide is applicable to constructs usedfor expression in yeast.

Efficient post-translational glycosylation and expression of recombinantpolypeptides can also be achieved in Baculovirus systems in insectcells, as are well known in the art.

In yet further embodiments, the peptides, polypeptides and/or fragmentsof this invention can be expressed in mammalian cells. Mammalian cellspermit the expression of peptides and polypeptides in an environmentthat favors important post-translational modifications such as foldingand cysteine pairing, addition of complex carbohydrate structures andsecretion of active protein. Vectors useful for the expression ofpeptides and polypeptides in mammalian cells are characterized byinsertion of the coding sequence between a strong (e.g., viral) promoterand a polyadenylation signal. The vectors can contain genes conferringeither, e.g., gentamicin or methotrexate resistance, for use asselectable markers. For example, the coding sequence can be introducedinto a Chinese hamster ovary (CHO) cell line using a methotrexateresistance-encoding vector. Presence of the vector RNA in transformedcells can be confirmed by Northern blot analysis and production of acDNA or opposite strand RNA corresponding to the polypeptide or fragmentcoding sequence can be confirmed by Southern and Northern blot analysis,respectively. A number of other suitable host cell lines capable ofproducing exogenous polypeptides have been developed in the art andinclude the CHO cell lines, HeLa cells, myeloma cell lines, Jurkat cellsand the like. Expression vectors for these cells can include expressioncontrol sequences, as described above.

The nucleic acids and/or vectors of this invention can be transferredinto the host cell by well-known methods, which vary depending on thetype of cell host. For example, calcium chloride transfection iscommonly used for prokaryotic cells, whereas calcium phosphate treatmentor electroporation can be used for other cell hosts.

The polypeptides, fragments, nucleic acids, vectors and cells of thisinvention can be present in a pharmaceutically acceptable carrier. By“pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the selected polypeptide,fragment, nucleic acid, vector or cell without causing substantialdeleterious biological effects or interacting in a deleterious mannerwith any of the other components of the composition in which it iscontained.

Furthermore, any of the compositions of this invention can comprise apharmaceutically acceptable carrier and a suitable adjuvant. As usedherein, “suitable adjuvant” describes an adjuvant capable of beingcombined with the polypeptide and/or fragment and/or nucleic acid ofthis invention to further enhance an immune response without deleteriouseffect on the subject or the cell of the subject. A suitable adjuvantcan be, but is not limited to, MONTANIDE ISA51 (Seppic, Inc., Fairfield,N.J.), SYNTEX adjuvant formulation 1 (SAF-1), composed of 5 percent(wt/vol) squalene (DASF, Parsippany, N.J.), 2.5 percent Pluronic, L121polymer (Aldrich Chemical, Milwaukee), and 0.2 percent polysorbate(Tween 80, Sigma) in phosphate-buffered saline. Other suitable adjuvantsare well known in the art and include QS-21, Freund's adjuvant (completeand incomplete), alum, aluminum phosphate, aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE) and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trealosedimycolate and cell wall skeleton (MPL+TDM+CWS) in 2% squalene/Tween 80emulsion.

The compositions of the present invention can also include othermedicinal agents, pharmaceutical agents, carriers, diluents,immunostimulatory cytokines, etc. Actual methods of preparing suchdosage forms are known, or will be apparent, to those skilled in thisart.

It is contemplated that the above-described compositions of thisinvention can be administered to a subject or to a cell of a subject toimpart a therapeutic benefit. Thus, the present invention furtherprovides a method of producing an immune response in a subject,comprising administering to the subject or to a cell of the subject aneffective amount of a polypeptide and/or biologically active fragment ofthis invention and/or a nucleic acid comprising a nucleotide sequenceencoding a polypeptide and/or biologically active fragment of thisinvention. The cell of the subject can be in vivo or ex vivo and can be,but is not limited to a CD8+T lymphocyte (e.g., a cytotoxic Tlymphocyte) or an MHC I-expressing antigen presenting cell, such as adendritic cell, a macrophage and/or a monocyte. Detection of an immuneresponse in the subject or in the cells of the subject can be carriedout according to methods standard in the art for detecting a humoraland/or cellular immune response.

Furthermore, the present invention provides a method of eliciting animmune response in a subject, comprising administering to the subject aneffective amount of a polypeptide and/of fragment of this invention.

Also provided herein is a method of eliciting an immune response in asubject, comprising administering to the subject an effective amount ofa nucleic acid and/or vector of this invention.

In additional embodiments, the present invention provides a method ofproviding passive immunity to a subject, comprising administering to thesubject an effective amount of an antibody of this invention to thesubject.

The compositions of this invention can also be employed as a therapeuticand/or prophylactic formulation and administered to a subject in needthereof. Thus, the present invention provides a method of treating orpreventing infection or intoxication by Mycoplasma pneumoniae in asubject, comprising administering to the subject an effective amount ofa polypeptide and/or fragment of this invention, a nucleic acid and/orvector of this invention, and/or an antibody of this invention.

In addition, the present invention provides a method of treating orpreventing infection or intoxication caused by Mycoplasma pneumoniae ina subject comprising contacting an immune cell of the subject with anyof the polypeptides, fragments, nucleic acids, vectors and/or antibodiesof this invention. The cell can be in vivo or ex vivo and can be, forexample, a CD8⁺T cell which is contacted with the polypeptide and/orfragment of this invention in the presence of a class I MHC molecule,which can be a soluble molecule or it can be present on the surface of acell which expresses class I MHC molecules. The cell can also be anantigen presenting cell or other class I MHC-expressing cell which canbe contacted with the nucleic acids and/or vectors of this inventionunder conditions whereby the nucleic acid or vector is introduced intothe cell by standard methods for uptake of nucleic acid and vectors. Thenucleic acid encoding the polypeptide and/or fragment of this inventionis then expressed and the polypeptide and/or fragment product isprocessed within the antigen presenting cell or other MHC I-expressingcell and presented on the cell surface as an MHC I/antigen complex. Theantigen presenting cell or other class I MHC-expressing cell is thencontacted with an immune cell of the subject which binds the class IMHC/antigen complex and elicits an immune response which treats orprevents Mycoplasma pneumoniae infection in the subject.

As set forth above, it is contemplated that in the methods wherein thecompositions of this invention are administered to a subject or to acell of a subject, such methods can further comprise the step ofadministering a suitable adjuvant to the subject or to a cell of thesubject. The adjuvant can be in the composition of this invention or theadjuvant can be in a separate composition comprising the suitableadjuvant and a pharmaceutically acceptable carrier. The adjuvant can beadministered prior to, simultaneous with, or after administration of thecomposition containing any of the polypeptides, fragments, nucleic acidsand/or vectors of this invention. For example, QS-21, similar to alum,complete Freund's adjuvant, SAF, etc., can be administered withindays/weeks/hours (before or after) of administration of the compositionof this invention. The effectiveness of an adjuvant can be determined bymeasuring the immune response directed against the polypeptide and/orfragment of this invention with and without the adjuvant, using standardprocedures, as described in the Examples herein and as are well known inthe art.

As set forth above, the subject of this invention can be any subject inneed of the immune response of this invention and/or in need oftreatment for or prevention from Mycoplasma pneumoniae infection, aswell as any subject in whom it is desirable to induce an immune responseto Mycoplasma pneumoniae. Symptoms of Mycoplasma pneumoniae infectioncan include tracheobronchitis and pneumonia with extrapulmonarypathologies, such as neurologic, cardiac, gastrointestinal,dermatologic, renal and joint complications. A range of serological(elevated IgM and IgG seroconversion) assays and PCR detection can beused for diagnosing M. pneumoniae infection. Appropriate treatment canlead to resolution of respiratory symptoms such as decreased fever andcough, complete recovery of respiratory function including normal lungradiogram, and normal levels of tissue enzymes and CSF analysis. Also,decreased levels of M. pneumoniae cells, antigens and nucleic acids inblood, sputum, bronchial lavage should accompany effective treatment.

Common sources of infection can include infected individuals coughing,sneezing and transmitting aerosols containing M. pneumoniae. Thetransmission rate is very high, which is why M. pneumoniae is such acommon cause of community acquired pneumonia. Highest targets ofinfection are children, especially 5-9 years old and adults between ages25-40, although infection can occur among all ‘healthy’ individuals.Thus, a subject for whom the methods of this invention would beindicated for preventing M. pneumoniae infection can, in someembodiments, be a child or young adult.

The compositions of this invention can be administered to a cell of asubject or to a subject either in vivo or ex vivo. For administration toa cell of the subject in vivo, as well as for administration to thesubject, the compositions of this invention can be administered orally,parenterally (e.g., intravenously), by intramuscular injection, byintraperitoneal injection, subcutaneous injection, transdermally,extracorporeally, topically or the like. Also, the compositions of thisinvention can be pulsed onto dendritic cells, which are isolated orgrown from a subject's cells, according to methods well known in theart, or onto bulk peripheral blood mononuclear cells (PBMC) or variouscell subfractions thereof from a subject.

The exact amount of the composition required will vary from subject tosubject, depending on the species, age, weight and general condition ofthe subject, the particular composition used, its mode of administrationand the like. Thus, it is not possible to specify an exact amount forevery composition of this invention. However, effective amount can bedetermined by one of ordinary skill in the art using only routineexperimentation given the teachings herein.

As an example, to a subject diagnosed with M. pneumoniae infection orknown to be at risk of being infected with M. pneumoniae or in whom itis desirable to induce an immune response to Mycoplasma pneumoniae,between about 50-1000 nM and more preferably, between about 100-500 nMof a polypeptide and/or biologically active fragment of this inventioncan be administered subcutaneously and can be in an adjuvant, at one tothree hour/day/week intervals until an evaluation of the subject'sclinical parameters indicate that the subject is not infected by M.pneumoniae and/or the subject demonstrates the desired immunologicalresponse. Alternatively, a polypeptide and/or fragment of this inventioncan be pulsed onto dendritic cells at a concentration of between about10-100 μM and the dendritic cells can be administered to the subjectintravenously at the same time intervals. The treatment can be continuedor resumed if the subject's clinical parameters indicate that M.pneumoniae infection is present and can be maintained until theinfection is no longer detected by these parameters and/or until thedesired immunological response is achieved.

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the subject's body according to standard protocolswell known in the art. The polypeptides and/or biologically activefragments of this invention can be introduced into the cells via knownmechanisms for uptake of polypeptides into cells (e.g., phagocytosis,pulsing onto class I MHC-expressing cells, liposomes, etc.). The cellscan then be infused (e.g., in a pharmaceutically acceptable carrier) ortransplanted back into the subject per standard methods for the cell ortissue type. Standard methods are known for transplantation or infusionof various cells into a subject.

The nucleic acids and vectors of this invention can also be administeredto a cell of the subject either in vivo or ex vivo. The cell can be anycell that can take up and express exogenous nucleic acid and produce thepolypeptides and/or fragments of this invention. In some embodiments,the polypeptides and/or fragments of this invention can be produced by acell that secretes them, whereby the polypeptide and/or fragment isproduced and secreted and then taken up and subsequently processed by anantigen presenting cell or other class I MHC-expressing cell andpresented to the immune system for induction of an immune response. Inother embodiments, the nucleic acids and/or vectors of this inventioncan be directly introduced into an antigen presenting cell and/or otherclass I MHC-expressing cell in which the polypeptide and/or fragment isproduced and processed directly and presented to the immune system onthe cell surface.

The nucleic acids and vectors of this invention can be administeredorally, intranasally, parenterally (e.g., intravenously), byintramuscular injection, by intraperitoneal injection, transdermally,extracorporeally, topically or the like. In the methods described hereinwhich include the administration and uptake of exogenous DNA into thecells of a subject (i.e., gene transduction or transfection), thenucleic acids of the present invention can be in the form of naked DNAor the nucleic acids can be in a vector for delivering the nucleic acidsto the cells for expression of the polypeptides and/or fragments of thisinvention. The vector can be a commercially available preparation or canbe constructed in the laboratory according to methods well known in theart.

Delivery of the nucleic acid or vector to cells can be via a variety ofmechanisms. As one example, delivery can be via a liposome, usingcommercially available liposome preparations such as LIPOFECTIN,LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen,Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison,Wis.), as well as other liposomes developed according to proceduresstandard in the art. In addition, the nucleic acid or vector of thisinvention can be delivered in vivo by electroporation, the technologyfor which is available from Genetronics, Inc. (San Diego, Calif.) aswell as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp.,Tucson, Ariz.).

As one example, vector delivery can be via a viral system, such as aretroviral vector system, which can package a recombinant retroviralgenome. The recombinant retrovirus can then be used to infect andthereby deliver to the infected cells nucleic acid encoding thepolypeptide and/or fragment of this invention. The exact method ofintroducing the exogenous nucleic acid into mammalian cells is, ofcourse, not limited to the use of retroviral vectors. Other techniquesare widely available for this procedure including the use of adenoviralvectors, alphaviral vectors, adeno-associated viral (AAV) vectors,lentiviral vectors, pseudotyped retroviral vectors and vaccinia viralvectors, as well as any other viral vectors now known or developed inthe future. Physical transduction techniques can also be used, such asliposome delivery and receptor-mediated and other endocytosismechanisms. This invention can be used in conjunction with any of theseor other commonly used gene transfer methods.

As another example, if the nucleic acid of this invention is deliveredto the cells of a subject in an adenovirus vector, the dosage foradministration of adenovirus to humans can range from about 10⁷ to 10⁹plaque forming units (pfu) per injection, but can be as high as 10¹²,10¹⁵ and/or 10²⁰ pfu per injection. Ideally, a subject will receive asingle injection. If additional injections are necessary, they can berepeated at daily/weekly/monthly intervals for an indefinite periodand/or until the efficacy of the treatment has been established. As setforth herein, the efficacy of treatment can be determined by evaluatingthe symptoms and clinical parameters described herein and/or bydetecting a desired immunological response.

The exact amount of the nucleic acid or vector required will vary fromsubject to subject, depending on the species, age, weight and generalcondition of the subject, the particular nucleic acid or vector used,its mode of administration and the like. Thus, it is not possible tospecify an exact amount for every nucleic acid or vector. However, anappropriate amount can be determined by one of ordinary skill in the artusing only routine experimentation given the teachings herein.

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The nucleic acids and vectors of this invention can beintroduced into the cells via any gene transfer mechanism, such as, forexample, virus-mediated gene delivery, calcium phosphate mediated genedelivery, electroporation, microinjection or proteoliposomes. Thetransduced cells can then be infused (e.g., in a pharmaceuticallyacceptable carrier) or transplanted back into the subject per standardmethods for the cell or tissue type. Standard methods are known fortransplantation or infusion of various cells into a subject.

Parenteral administration of the peptides, polypeptides, nucleic acidsand/or vectors of the present invention, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. As used herein, “parenteral administration” includesintradermal, intranasal, subcutaneous, intramuscular, intraperitoneal,intravenous and intratracheal routes, as well as a slow release orsustained release system such that a constant dosage is maintained. See,e.g., U.S. Pat. No. 3,610,795, which is incorporated by reference hereinin its entirety.

The efficacy of treating or preventing Mycoplasma pneumoniae infectionby the methods of the present invention can be determined by detecting aclinical improvement as indicated by a change in the subject's symptomsand/or clinical parameters, as would be well known to one of skill inthe art.

It is further contemplated that the compositions of the presentinvention can be used in diagnostic and therapeutic applications. Thus,the present invention provides a method of detecting the presence of apolypeptide and/or fragment of this invention in a sample, comprisingcontacting the sample with an antibody of this invention underconditions whereby an antigen/antibody complex can form and detectingformation of an antigen/antibody complex, thereby detecting the presenceof a Mycoplasma pneumoniae polypeptide and/or fragment of this inventionin the sample.

Additionally, the present invention provides a method of detecting thepresence of an antibody of this invention in a sample, comprisingcontacting the sample with a polypeptide and/or fragment of thisinvention under conditions whereby an antigen/antibody complex can formand detecting formation of an antigen/antibody complex, therebydetecting the presence of a Mycoplasma pneumoniae antibody of thisinvention in the sample.

The sample of this invention can be any sample in which Mycoplasmapneumoniae exotoxin can be present. For example, the sample can be abody fluid, cells or tissue that can contain Mycoplasma pneumoniaeexotoxin, including but not limited to, blood, serum, plasma, saliva,sputum, bronchoalveolar lavage, urine, semen, joint fluid, cerebrospinalfluid and cells, fluids and/or tissue from all organs to which CARDStoxin can disseminate including lung, liver, heart, brain, kidney,spleen, muscle, etc.

Additionally, the present invention provides a method of diagnosingMycoplasma pneumoniae infection in a subject comprising contacting abiological sample from the subject with a polypeptide and/or fragment ofthis invention under conditions whereby an antigen/antibody complex canform; and detecting formation of an antigen/antibody complex, therebydiagnosing Mycoplasma pneumoniae infection in the subject.

A method of diagnosing Mycoplasma pneumoniae infection in a subject isfurther provided, comprising contacting a biological sample from thesubject with an antibody of this invention under conditions whereby anantigen/antibody complex can form; and detecting formation of anantigen/antibody complex, thereby diagnosing Mycoplasma pneumoniaeinfection in the subject.

In further embodiments, the present invention provides a method ofdiagnosing infection by Mycoplasma pneumoniae in a subject, comprisingcontacting a biological sample from the subject with the nucleic acid ofthis invention under conditions whereby hybridization of nucleic acidmolecules can occur and detecting a hybridization complex, therebydiagnosing infection by Mycoplasma pneumoniae in the subject.

In additional embodiments, the present invention provides a method ofidentifying a subject infected with Mycoplasma pneumoniae as having apoor prognosis, comprising:

a) establishing a correlation between the presence of and/or an amountof a polypeptide, fragment, nucleic acid and/or antibody of thisinvention in a sample of test subjects infected with Mycoplasmapneumoniae and who have or had a poor prognosis;

b) detecting in a biological sample from the subject the presence ofand/or an amount of the polypeptide, fragment, nucleic acid and/orantibody of this invention correlated with a poor prognosis, therebyidentifying the subject infected with Mycoplasma pneumoniae as having apoor prognosis. For example, a correlation can be made between a levelof antibodies to the CARDS toxin and a degree of respiratory and/orpulmonary dysfunction indicative of a poor prognosis.

The present invention also provides various screening assays that employthe polypeptides, fragments and/or nucleic acids of this invention. Inparticular, provided herein is a method of identifying a substancehaving the ability to inhibit or enhance the binding activity of apolypeptide and/or biologically active fragment of this inventioncomprising contacting the substance with the CARDS protein or abiologically active fragment thereof under conditions whereby bindingcan occur and detecting a decrease or increase in the amount of bindingin the presence of the substance as compared to a control amount ofbinding in the absence of the substance, thereby identifying a substancehaving the ability to inhibit or enhance the binding activity of theCARDS toxin.

Inhibition or enhancement of binding activity can be detected by any ofa variety of art-recognized methods for evaluating binding activity. Asone example, the substance to be tested and the CARDS polypeptide and/orfragment can be contacted in the presence of target cells or a targetsubstrate (e.g., surfactant protein A; SP-A) known to bind thepolypeptide or fragment. The amount of binding of polypeptide orfragment to the cells or the substrate in the presence of the substanceand the amount of binding of polypeptide or fragment to the cells or thesubstrate in the absence of the substance is determined and a decreaseor increase in the amount of binding in the presence of the substanceidentifies the substance as having the ability to inhibit or enhancebinding.

In some embodiments, binding of polypeptide and/or fragment to targetcells or a target substrate can be measured by attaching a detectablemoiety to the polypeptide or fragment (e.g., a fluorescence moiety,histochemically detectable moiety, radioactive moiety, etc.). The amountof detectable moiety can be measured in the presence and absence of thesubstance to be tested and the amounts can be compared to determineinhibition or enhancement. Binding activity can also be determined bycomparing the amount of cytopathology observed in a monolayer of targetcells in the presence and absence of the substance to be tested. Targetcells that can be used in such a binding assay include, but are notlimited to, Chinese hamster ovary (CHO) cells, Hep2 cells, human lungand kidney epithelial and fibroblast cells, and any other mammaliancells that exhibit sensitivity to CARDS toxin now known or lateridentified.

In addition, the present invention provides a method of identifying asubstance having the ability to inhibit or enhance the translocatingactivity of a polypeptide and/or a biologically active fragment of thisinvention, comprising contacting the substance with the polypeptide ofthis invention and/or a biologically active fragment thereof underconditions whereby translocation activity can occur and detecting adecrease or increase in the amount of translocation activity in thepresence of the substance as compared to a control amount oftranslocation activity in the absence of the substance, therebyidentifying a substance having the ability to inhibit or enhance thetranslocating activity of the CARDS toxin.

Inhibition or enhancement of translocating activity can be detected byany of a variety of art-recognized methods for evaluating translocatingactivity. As one example, the substance to be tested and the CARDSpolypeptide and/or fragment can be contacted in the presence of targetcells known to translocate the CARDS exotoxin. The amount oftranslocation of polypeptide or fragment into the cells in the presenceof the substance and the amount of translocation of polypeptide orfragment into the cells in the absence of the substance is determinedand a decrease or increase in the amount of translocation in thepresence of the substance identifies the substance as having the abilityto inhibit or enhance translocation of the CARDS exotoxin. Translocationof polypeptide and/or fragment into target cells can be measured byattaching a detectable moiety to the polypeptide or fragment (e.g., afluorescence moiety, histochemically detectable moiety, radioactivemoiety, etc.). The amount of translocated detectable moiety can bemeasured in the presence and absence of the substance to be tested andthe amounts can be compared to determine inhibition or enhancement oftranslocation. Translocation activity can also be determined bycomparing the amount of cytopathology observed in a monolayer of targetcells in the presence and absence of the substance to be tested. Targetcells that can be used in such a translocation assay include, but arenot limited to, Chinese hamster ovary (CHO) cells, etc.

Further provided is a method of identifying a substance having theability to enhance or inhibit the immunogenic activity of the CARDStoxin of this invention and/or a biologically active fragment thereof,comprising contacting the substance with the CARDS toxin or animmunogenic fragment thereof under conditions whereby a measurableimmune response can be elicited and detecting an increase or decrease inthe amount of immune response in the presence of the substance, ascompared to a control amount of immune response in the absence of thesubstance, thereby identifying a substance having the ability to enhanceor inhibit immunogenic activity of the CARDS toxin. Assays to detect andmeasure immune responses are well known in the art and can be employedto detect either humoral or cellular immune responses.

In additional embodiments, the present invention provides a method ofidentifying a substance having the ability to inhibit or enhance theADP-ribosylating activity of the CARDS toxin of this invention and/orbiologically active fragments thereof, comprising contacting thesubstance with the CARDS toxin or biologically active fragment thereofunder conditions whereby ADP ribosylation can occur and detecting adecrease or increase in the amount of ADP ribosylation in the presenceof the substance as compared to a control amount of ADP ribosylation inthe absence of the substance, thereby identifying a substance having theability to inhibit or enhance the ADP ribosylating activity of the CARDStoxin.

Methods for detecting ADP ribosylating activity are well known in theart and are described, for example, in the Examples section providedherein.

Further provided is a method of identifying a substance having theability to inhibit or enhance the cytopathology-inducing activity of theCARDS toxin of this invention and/or a biologically active fragmentthereof, comprising contacting the substance with the CARDS toxin orbiologically active fragment thereof under conditions wherebycytopathology (e.g., changes in cell morphology, monolayercharacteristics, etc.) of target cells can be induced and detecting adecrease or increase in the amount of cytopathology in the presence ofthe substance, as compared to a control amount of cytopathology in theabsence of the substance, thereby identifying a substance having theability to inhibit or enhance the cytopathology-inducing activity of theCARDS toxin or biologically active fragment thereof.

Methods of detecting cytopathology of cells are well known in the artand are described, for example, in the Examples section herein.

Substances identified in the screening assays of this invention to havethe ability to inhibit or enhance various of the activities of thepolypeptides and/or fragments of this invention can be employed inmethods of diagnosing M. pneumoniae infection, as well as in methods oftreating and/or preventing M. pneumoniae infection. For example, suchsubstances can be present in a pharmaceutically acceptable carrier foradministration to a subject and an effective amount of the substance canbe administered to a subject to treat and/or prevent infection byMycoplasma pneumoniae.

It is also contemplated that the present invention includes methods ofscreening Mycoplasma pneumoniae cultures for mutants defective in one ormore of the biological activities of the CARDS exotoxin, for use in avaccine preparation. Such mutants can be identified as having a defectin any of the biological activities of the CARDS exotoxin according tothe protocols described herein and as are known in the art. Such mutantscan be further tested for being attenuated in the ability to produce aclinical infection in a subject (i.e., for virulence potential) and thenfurther evaluated for use as a vaccine according to known protocols.

For example, in one embodiment, CARDS toxin mutants of Mycoplasmapneumoniae (e.g., having a mutation in the CARDS coding sequence orlacking the CARDS coding sequence) can be generated through suchart-known techniques as gene disruption and their virulence potentialdeter mined by challenge studies in hamsters and by adherence andcytopathology assessments in hamster tracheal rings in organ culture andin cell culture, as is well known in the art. In addition,complementation studies can be performed to restore the defectiveactivity of the CARDS toxin, in order to characterize the mutant.

The present invention is more particularly described in the followingexamples, which are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

EXAMPLES Mycoplasma Strains and DNA Isolation Conditions

M. pneumoniae reference strain M129/B9 and clinical isolates S1, L2, JL1and RJL1 were grown to late logarithmic phase in SP-4 medium at 37° C.for 72 h in 150-cm² tissue culture flasks. Mycoplasmas were harvested bywashing three times with PBS [150 mM NaCl, 10 mM sodium phosphate, pH7.4] and pelleting at 12,500 g for 15 min at 4° C. M. pneumoniaechromosomal DNA was isolated using Easy DNA kit according to themanufacturer's protocol (Invitrogen).

Mycoplasma Culture Conditions for Radiolabeling.

Wild-type Mycoplasma pneumoniae M129/B9 and clinical isolates were grownin SP-4 medium as above. Mycoplasma monolayers in logarithmic growthphase were washed two times with 10 ml PBS (pH 7.4) and one time withDulbecco Modified Eagle Medium (DMEM) without L-cysteine andL-methionine and resuspended in 10 ml Dulbecco Modified Eagle Medium(DMEM) without L-cysteine and L-methionine supplemented with 10%heat-inactivated fetal bovine serum and 100 μCi L-[³⁵S]methionine. After4 h incubation at 37° C., supernatants were removed and monolayerswashed twice with 25 ml PBS. Mycoplasma cells were scraped into a volumeof 10 ml sterile PBS, collected by centrifugation at 9,500×g and washedmultiple times in PBS. Cell pellets were resuspended in 1 ml completelysis buffer (CLB) prepared shortly before use (150 mM NaCl, 10 mM Tris,20 μM EGTA, 0.5 M Triton-X 114, 1 mM CaCl₂ and protease inhibitors 1 μMpepstatin A, 200 μM PMSF, 1 mM N-α-p-tosyl-L-lysine chloromethyl ketone(TLCK), and 10 μM leupeptin. Cell pellets in CLB were sheared through 25gauge needles using 3 ml syringes to obtain clear lysis. 20 μl aliquotsof resuspended cell lysate were transferred to separate microfuge tubesfor SDS-PAGE analysis and scintillation counter assessment (BeckmanInstruments Inc. Irvine, Calif.). Radiolabeled lysates were diluted to 6ml in CLB and passed through control and experimental SP-A columns (seebelow) in parallel.

Purification of SP-A Binding Proteins

A 20×1.2 cm control glass column was packed with 3 ml uncoupledSepharose, another identical (experimental) column was packed with 3 mlSepharose coupled to SP-A Coupling of SP-A to Sepharose CL-4B wasperformed as follows: A total of 1.5 mg of SP-A was coupled to 2 g ofCNBr-activated Sepharose CL-4B according to the manufacture'sinstructions except the coupling buffer was 10 mM sodium bicarbonate, pH8.3. SP-A coupled Sepharose was stored in 5 ml of 5 mM Tris pH 7.5,containing 1 mM NaN₃. Columns were equilibrated with 50 ml CLB prior toaddition of radiolabeled cell lysates. Radiolabeled cell lysates werecollected and reapplied to each column 3-4 times. After samples wereadded, columns were washed with 10 times volume of packed material toremove unbound proteins. M. pneumoniae SP-A-binding proteins were elutedusing a NaCl gradient (0.2 to 3 M NaCl) containing 10 mM EDTA. Eluateswere collected as 1 ml fractions, and 20 d from each fraction wasassayed for specific activity with a scintillation counter.

SDS-PAGE and Autoradiogram.

Fractions eluted from columns were individually dialyzed/desaltedagainst PBS and concentrated by an Amicon concentrator/lyophilizer to1/30^(th) of original volume. Samples were resolved in 12% SDS-PAGE andstained with Coomassie brilliant blue or transferred to nitrocelluloseand exposed to Kodak XRP-40 x-ray film (Kodak, Rochester, N.Y.) for 4-8days.

MALDI-TOF Protein Sequencing.

SDS-polyacrylamide gels containing M. pneumoniae SP-A binding proteinswere stained with Coomassie brilliant blue and washed thoroughly indistilled water.

Individual protein bands were excised from acrylamide gels and subjectedto MALDI-TOF by the microsequencing facility at Baylor College ofMedicine (Houston, Tex.).

Bacterial Strains, Plasmids and DNA Manipulations.

Escherichia coli INVαF′ [F′ endA1rec1hsdR17supE44gyrA96lacZM15 (lacZYAargF)] (Invitrogen) and E. coli BL21(DE3) [F′ ompT hsdS (r_(B) ⁻m_(B)⁻) gal dcm λ(DE3) pLysS] were grown in Luria Bertani (LB) broth and usedto clone and express mycoplasma CARDS toxin genes. For DNAmanipulations, the following vectors were used: pCR2.1 (Ap^(r), Km^(r)TA cloning vector [Invitrogen]) and pET19b (Ap^(r), N-terminal His¹⁰tag, expression vector [Novagen]). Plasmid DNA was purified using theQIAprep spin protocol according to the manufacturer (Qiagen).

SOE-PCR

In attempting to determine precise binding motifs of M. pneumoniae SP-Abinding proteins, both full size and truncated overexpressed proteinsare employed. Initially, the number of truncated proteins will dependupon the number and location of UGA codons. Should the possibility arisethat SP-A binding motifs are located in UGA-coded regions of a protein,this issue will be addressed using full-size proteins, orprotease-digested peptide fragments, or synthetic peptides as describedherein. UGA usage problems in genes encoding SP-A binding proteins, aswell as other mycoplasma proteins, are known. In such proteins, the UGAcodons in the corresponding genes are modified by site-directedmutagenesis to express full size proteins. PCR-based “splicing byoverlap extension” (SOE) methods are employed to mutagenize UGA in thesegenes. This method is based on the principle that two overlappingcomplementary ends may prime on each other and be extended to yield ahybrid product, and a second PCR with two primers annealing at thenon-overlapping ends will amplify this hybrid. An example of a stepwisestrategy for SOE-PCR is as follows. 1. ‘a’ and ‘d’ are primers for agene and ‘b’ and ‘c’ are primers to mutagenize the UGA region. 2.Amplification carried out with primers ‘a’ and ‘b’ and using genomic DNAas template gives a DNA fragment “AB” of the gene. 3. Amplificationcarried out with primers ‘c’ and ‘d’ and using genomic DNA as templatewill give DNA fragment “CD” of the gene. 4. Amplification with primers‘a’ and ‘d’ and using DNA fragments “AB”+“CD” as templates will give theUGA modified mutant gene fragment. The overlapping primers covering theUGA codon in the genes are modified as UGG, a codon that still codes fortryptophan, and the primer sets depend upon the number of UGAs to bemutated in each gene. In all cases, genomic DNA of M. pneumoniae is usedas template, and AccuTaq polymerase mix (Sigma) is used to amplify DNAfragments.

Immunoblot Assay

Mycoplasma total proteins or purified recombinant CARDS protein wereresolved on 4-12% SDS-polyacrylamide gels (NuPAGE, InVitrogen) (His-tagreleased, i.e., minus His tag) and transferred electrophoretically tonitrocellulose membranes (Towbin et al., 1979). Membranes were blockedfor two hours with 5% (wt/vol) blotto [nonfat dry milk in TBS containing0.1% Tween-20 (TBST)], followed by three washes with TBST, and incubatedwith M. pneumoniae infected patient sera (1:50 to 1:100 in 2% blotto) atRT for 2 h. Then, individual membranes were washed three times (15 minper wash) in TBST and incubated for 2 h (ambient temperature) withalkaline phosphatase-conjugated goat anti-human IgG Abs at a dilution of1:2000 in TBST, which were washed 5 additional times with TBST, thencolor developed with BCIP/NBT tablets (Sigma).

FIG. 1 is an immunoblot of sera from three patients, RJ, 1970 and MJ,infected with Mycoplasma pneumoniae. Purified M. pneumoniae recombinantCARDS toxin was resolved in 4-12% SDS-PAGE and transferred tonitrocellulose membranes. Membranes were blocked for two hours with 5%blotto and treated with patients' sera for two hours at roomtemperature. Patients' sera were diluted as follows. RJ and MJ: 1:50,and 1970:1:100 in 2% blotto. Membranes were washed and treated withalkaline phosphatase-conjugated goat anti-human antibodies diluted1:2000 in TBS-T and two hours and color developed. Patients RJ and MJdied within about three weeks of infection and patient 1970 washospitalized with mycoplasmal pneumonia and recovered. A 68 kDa MWrecombinant CARDS toxin is indicated by the arrow; higher molecularweight and diffuse bands represent His-tagged subpopulation ofrecombinant CARDS toxin. Detection of antibodies to the CARDS toxinindicates in situ synthesis of CARDS toxin during infection and itsimmunogenicity.

Additional Studies on Patients Infected with M. Pneumoniae.

In further studies, acute and convalescent sera were collected frompatients with Mycoplasma pneumoniae-diagnosed respiratory infectionsthat ranged from tracheobronchitis to bronchopneumonia. Two or threeblood samples were obtained from each patient. The first blood samplewas collected during the acute phase of the disease, approximately twoweeks following exposure to M. pneumoniae. The second and third“convalescent” serum samples were obtained 14 and 28 days later,respectively. Control baseline serum samples were obtained from pregnantwomen attending the University of Texas Health Science Center at SanAntonio OB-GYN clinic.

All serum samples were assessed by immunoblotting against total M.pneumoniae proteins. Specifically, to detect CARDS toxin protein inpatients' sera, M. pneumoniae total cell preparations of differentclinical isolated (RJ1, J1, S1 and L2) and laboratory strain (B9) weredissolved in 1500 SDS sample buffer, boiled for two minutes andseparated by SDS-PAGE using 4-12% NuPAGE SDS-polyacrylamide gels.Proteins were transferred to nitrocellulose membranes (Shleicher &Schull, Dassel, Germany) by electroblotting. Membranes were blocked forone hour at room temperature with blocking buffer (20 mM Tris-base, 150mM NaCl, 3% skim milk powder) and incubated with anti-CARDS Toxin mousepolyclonal antibodies diluted 1:2000 in antibody buffer (20 mMTris-base, 150 mM NaCl, 3% skim milk powder) for one hour at 37° C.Bound IgG was detected with alkaline phosphatase (AP)-conjugatedgoat-antimouse IgG diluted 1:3000. Membranes were developed for 1-5minutes with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate p-toluidium (BCIP) solution. Results of the immunoblottingshow a colored band of 68 kDa molecular weight on each membrane and thusdemonstrate the presence of the CARDS toxin protein in each clinicalisolate at concentrations that appear to vary among individuals.

Additional immunoblot analyses were carried out to detect antibodies toCARDS toxin in infected patients' sera wherein M. pneumoniae recombinant68 kDa CARDS (rCARDS) toxin (3 μg) or the N terminal domain of CARDStoxin, rD1 (1 μg) as described herein was dissolved in 150 μl LDS samplebuffer (NuPAGE), boiled for two minutes and separated by SDS-PAGE using4-12% NuPAGE SDS-polyacrylamide gels. Proteins were transferred tonitrocellulose membranes (Schleicher & Schull, Dassel, Germany) byelectroblotting and membranes were blocked for one hour at roomtemperature with blocking buffer (20 mM Tris-base, 150 mM NaCl, 3% skimmilk powder). Membranes were cut into 3 mm strips and incubated withhuman serum samples diluted 1:200 in buffer (20 mM Tris-base, 150 mMNaCl, 3% skim milk powder) for one hour at 37° C. Serum samples werefrom M. pneumonia-infected patients designated patients 1 and 2 and thefirst serum samples were collected during the acute phase of disease(designated 1-1 and 2-1, respectively). The second serum samples (1-2and 2-2) and third serum samples (1-3 and 2-3) were obtained 14 and 28days later, respectively.

Bound IgG was detected with alkaline phosphatase (AP)-conjugatedgoat-antihuman IgG diluted 1:3000. Individual strips were developed for1-5 minutes with nitroblue tetrazolium (NBT) and5-bromo-4-chloro-3-indolyl phosphate p-toluidium (BCIP) solution.Results of the immunoblotting showed a colored band of 68 kDa molecularweight on each membrane containing rCARDs toxin and colored bands of 32kDa and 28 kDa on each membrane containing the D1 domain, thusdemonstrating seroconversion in these patients and detection ofantibodies to the CARDS toxin, either as a recombinant protein or as theD1 domain. In the latter assay, the color intensity of each band appearsto increase in the samples in a manner consistent with the time courseof collection from the patient during the course of the disease (i.e.,1-1<1-2<1-3) (FIG. 3).

ELISAs were also carried out on the samples collected from patients 1and 2 described above (i.e., samples 1-1, 1-2, 1-3, 2-1, 2-2, and 2-3).In these assays, washing at each stage was performed at least threetimes with PBS and sera and antibodies were diluted in 1% BSA in PBS.Each well of Immulon 4 HBX Immunoplates (Dynox) was coated overnight at4° C. with 50 μl of rCARDS toxin/D1 (1 μg/well) diluted incarbonate/bicarbonate buffer (32 mM Na₂CO₃, 64 mM NaHCO₃). Individualplates were washed, 100 μl of 1 mg/ml (wt/vol) BSA in PBS was added toeach well, and incubation continued for two hours at room temperature.After washing, 50 μl of diluted human serum samples (1/50 to 1/3200)were added to each well, and plates were incubated for two hours at roomtemperature. Then, plates were washed, and 50 μl of diluted (1:1000)alkaline phosphatase (AP)-conjugated goat-antihuman IgG (Zymed) wereadded to each well. Plates were incubated for 1.5 hours at roomtemperature, washed and 50 μl of substrate solution [p-nitrophenylphosphate (PNPP)/0.1M Tris pH 9.6] was added and plates were incubatedat room temperature for 30-60 minutes. Absorbance values at 450 nm weredetermined for each well.

The results for patient 1 with serum dilutions of 1/100 and 1/200 andrD1 as the antigen showed a decrease in optical density at the greaterdilution of serum and a stepwise increase in optical density in thesamples collected sequentially during the course of disease (i.e.,1-1<1-2<1-3) (FIG. 3A). This stepwise increase correlates with theincreased color intensity observed with these serum samples in theimmunoblot assay (FIG. 3A). Similar results were obtained withsequential serum samples from patient 1 when rCARDS Toxin was used asthe antigen.

The results for patient 2 with serum dilutions of 1/100, 1/200, 1/400,1/800, 1/1600 and 1/3200 and rD1 as the antigen showed a decrease inoptical density as the dilution of serum increased and a stepwiseincrease in optical density in the samples collected sequentially duringthe course of disease (i.e., 2-1<2-2<2-3) (FIG. 3B). This stepwiseincrease correlates with the increased color intensity observed withthese serum samples in the immunoblot assay (FIG. 3B). Similar resultswere obtained with sequential serum samples from patient 2 when rCARDSToxin was used as the antigen.

Additional studies were conducted wherein each well of an Immulon 4 HBXImmunoplate (Dynox) was coated overnight at 4° C. with 50 μl of rCARDStoxin (1, 2 or 3 μg/well) diluted in carbonate/bicarbonate buffer. Afterwashing, 50 μl of diluted human serum samples (1/200 dilution ofconvalescent serum 1-3 as described above) was added to each well andplates were incubated for two hours at room temperature prior todetection of bound IgG. Negative patient serum control was alsoincluded. The results showed an optical density around 1.8 and 1.9±SEfor all three concentrations of rCARDS toxin and an optical density ofthe negative control around 0.6 and 0.7±SE for all concentrations oftoxin.

A further study was carried out as described above, except that eachwell of Immulon 4 HBX Immunoplates (Dynox) was coated overnight at 4° C.with 50 μl of CARDS rD1 domain diluted as follows: 1, 2, 3, 4, 5 or 6μg/well, in carbonate-bicarbonate buffer. Negative patient serum controlwas also included. The results show an optical density between 1.0 and1.2±SE for all six concentrations of rD1 domain and an optical densityof the negative control of 0.2±SE or less for all concentrations of rD1.

Overall, these immunoblot and ELISA studies demonstrate that both CARDStoxin and antibodies to CARDS toxin can be detected according to themethods of this invention and that the assays can be performed with aslittle as 1 μg of toxin either as the recombinant protein or as the D1domain. These studies also indicate that the D1 domain may be a bettertarget in an ELISA format, with lower background levels.

Identification of Mycoplasma pneumoniae by PCR in Sputum Samples UsingCARDS Toxin as a Target DNA Molecule

In this assay, phosphate buffered saline (PBS), with and without saliva,was mixed with a cell suspension (cells grown 2-3 days at 37° C. in SP-4medium; total cells ˜1×10⁹) of M. pneumoniae S1 cells in a 1:1 ratio andcentrifuged. The pellet was resuspended in 200 μl of water and incubatedat 4° C. for 20 minutes. The sample was then boiled at 100° C. for 15minutes. 37-40 μl of this sample was used for PCR in a total reactionvolume of 50 μl. The samples were serially diluted 10⁻¹ to 10⁻⁹ in PBS.PCR conditions were 95° C. for five minutes; 94° C. for one minute; 55°C. for one minute; 72° C. for one minute and 72° C. for 10 minutes, for30 cycles. The amplification primer set was Primer 12a forward: (nts.1197-1220; 24 bp) 5′ gcttgttctggaataccaagagtg 3′(SEQ ID NO:23) andPrimer 15a reverse: (nts. 1541-1564; 24 bp) 5′ ccattctacccaatcccagctgta3′ (SEQ ID NO:26). The product size of the amplicon was 368 base pairs.Detection was by ethidium bromide staining or autoradiography with a³²P-labeled probe. The probe used to detect the amplicon byautoradiography was Primer 14a forward: (nts 1371-1429; 59 bp) 5′gctggtattggaggggttattactataccccacaattaagtggttggtcttatcagatg 3′ (SEQ IDNO:25). Results of this study demonstrate that M. pneumoniae nucleicacid can be detected in the presence or absence of saliva and that onemycoplasma cell can be identified using this primer/probe set.

Cloning and Sequencing of CARDS

Based on the published genome sequence of M. pneumoniae M129/B9(Himmelreich et al., 1996, SEQ ID NO:7), the complete open reading frameof cards was analyzed. Translation of nucleotide sequences to aminoacids revealed the existence of eight TGA codons within the codingregion of cards. Start and stop codons and the eight intervening TGAcodons are indicated in bolded text.

tttttaattt gtaaaatttc attttttaaa aatgccaaat cctgttagat ttgtttaccgtgttgatttg agaagccctg aagaaatttt tgaacatggc ttttcaactt taggtgatgtgagaaatttc tttgaacaca ttctctccac taattttggt agaagctatt ttatttccacttcagaaaca cccacagcag ctattcgctt ctttggtagc tggttacggg aatatgtaccagagcacccc agaagggctt acttatatga aattcgtgcc gaccaacact tttacaatgcccgcgccact ggggagaact tgttagattt aatgcgtcaa agacaagtag tatttgactctggtgatcga gaaatggcac aaatgggaat tagagcttta cgcacttcct ttgcgtatcaacgtgaatgg tttaccgatg gtccaattgc agcagctaat gtccgtagtg cttgactagtagatgctgtt cccgttgaac ctggtcatgc tcaccacccg gctggtcgtg ttgtagagactactagaatt aatgaaccgg aaatgcacaa ccctcattat caagagctgc aaacccaagccaatgatcaa ccatgattgc caacaccagg aatagctact cctgtacatt tatcaattccccaagcagct tccgttgctg atgtttcgga aggtacttcc gcttcgctat cgtttgcgtgccctgattga agtccacctt ctagtaatgg tgaaaatccg ctagacaaat gcattgcggaaaagattgat aactataacc tacaatcctt accacagtac gctagcagtg taaaggaactggaagataca ccagtatacc taaggggaat taaaacgcaa aaaaccttta tgttacaagcagatccgcaa aataacaatg tctttttggt cgaagtaaac cccaaacaaa agtccagctttccccaaacc atcttctttt gggatgttta tcaacgaatt tgtctcaagg atttaactggtgcacaaatc agtctttcgc ttactgcctt tactactcag tatgctggtc agctcaaagtgcaccttagt gttagcgcgg ttaatgccgt gaaccaaaag tgaaaaatga caccgcaagacattgcaata actcagtttc gggtctcctc tgaactgtta ggtcaaactg aaaatggcttgttctgaaat accaagagtg gtggttcaca acacgatttg tatgtatgtc ctttgaaaaatccacctagt gatttggaag aattacaaat aattgttgat gaatgtacta cccatgcgcagtttgttact atgcgtgcag ctagcacctt ctttgttgat gttcagctag gctggtattgaaggggttat tactataccc cacaattaag tggttgatct tatcagatga aaacaccagatggacagata ttctatgatc taaaaacttc gaaaatcttc tttgtccagg acaaccaaaacgtgttcttt ctccataata aactcaacaa acaaactggt tacagctggg attgagtagaatggctaaaa catgacatga atgaggacaa agacgaaaac tttaaatggt acttttcgcgtgatgacctt accattcctt ccgttgaagg gcttaacttc cgccacattc gctgttacgctgacaaccag cagttaaagg tgatcataag cggttcacgt tggggcggtt ggtactccacttacgataaa gttgaaagta atgtcgaaga taagattttg gtcaaagatg gttttgatcgcttttagcga ttaagcttta acgtcactgt tttgctctaa tgttagaagc aaagatcttg

The entire cards sequence was amplified using forward primer5′-tttttacatatgccaaatcctgtt-3′ (primer 1, SEQ ID NO:12) and reverseprimer 5′-gatcgcttttagcgaggatcctttaacg-3′ (primer 2, SEQ ID NO:64),which produces NdeI and BamHI (underlined) sites at 5′ and 3′ ends ofthe cards ORF, respectively. Both fragments were ligated into the pCR2.1 vector and transformed into E. coli cells for automated sequencingusing M13 forward and reverse primers.

Site-directed mutagenesis of the cards gene to permit expression oftotal recombinant CARDS protein was necessary, which required thecorrection of TGAs to TGGs in order to encode tryptophan in E. coli.Therefore, specific primers were designed as indicated below. Primersbelow are also used to generate specific CARDS domains for generatingspecific antibody probes.

CARDS: Oligonucleotide Sequences within Selected (Above) NucleotideSequence.

Pri 1-16: Modified oligonucleotide sequence* to amplify the cardssequence. * modified nucleotides are given in bold. Complementaryoligonucleotide sequence are given underneath the reverse primers (2, 3,5, 7, 9, 11, 13 and 15)

Sequence of M. Pneumoniae CARDS.

The cards gene of M. pneumoniae reference strain M129/B9 and clinicalisolates (S1, L2, JL and RJL1) were cloned in a PCRII vectorindividually and sequenced.

M129/B9 represents the reference strain and S1, L2, RJL1 and JL areclinical isolates from patients in San Antonio and Dallas.

All clinical isolates have the same mutation at nucleotide 1112^((T→G))from the ATG start codon, which differs from the published referencestrain. However, in clinical isolate S1 three additional nucleotidechanges occur at nucleotide base positions 113^((T→C)), 922^((T→C)) and1172^((T→C)).

The following nucleotide changes were detected in the other clinicalisolates:

L2: 734^((A→G)) and 1112^((T→G)). JL: 1112^((T→G)). RJL1: 1112^((T→G))and 1174^((T→C)).

Coding Sequence of S1 (Mycoplasma pneumoniae Clinical Isolate)

Bolded gs shown were introduced by site directed mutagenesis in order toexpress CARDS protein in E. coli.

S1 Nucleotide sequence (SEQ ID NO: 8) atgccaaatc ctgttagatt tgtttaccgtgttgatttga gaagccctga agaaattttt   60 gaacatggct tttcaacttt aggtgatgtgagaaatttct ttgaacacat tccctccact  120 aattttggta gaagctattt tatttccacttcagaaacac ccacagcagc tattcgcttc  180 tttggtagct ggttacggga atatgtaccagagcacccca gaagggctta cttatatgaa  240 attcgtgccg accaacactt ttacaatgcccgcgccactg gggagaactt gttagattta  300 atgcgtcaaa gacaagtagt atttgactctggtgatcgag aaatggcaca aatgggaatt  360 agagctttac gcacttcctt tgcgtatcaacgtgaatggt ttaccgatgg tccaattgca  420 gcagctaatg tccgtagtgc ttggctagtagatgctgttc ccgttgaacc tggtcatgct  480 caccacccgg ctggtcgtgt tgtagagactactagaatta atgaaccgga aatgcacaac  540 cctcattatc aagagctgca aacccaagccaatgatcaac catggttgcc aacaccagga  600 atagctactc ctgtacattt atcaattccccaagcagctt ccgttgctga tgtttcggaa  660 ggtacttccg cttcgctatc gtttgcgtgccctgattgga gtccaccttc tagtaatggt  720 gaaaatccgc tagacaaatg cattgcggaaaagattgata actataacct acaatcctta  780 ccacagtacg ctagcagtgt aaaggaactggaagatacac cagtatacct aaggggaatt  840 aaaacgcaaa aaacctttat gttacaagcagatccgcaaa ataacaatgt ctttttggtc  900 gaagtaaacc ccaaacaaaa gcccagctttccccaaacca tcttcttttg ggatgtttat  960 caacgaattt gtctcaagga tttaactggtgcacaaatca gtctttcgct tactgccttt 1020 actactcagt atgctggtca gctcaaagtgcaccttagtg ttagcgcggt taatgccgtg 1080 aaccaaaagt ggaaaatgac accgcaagacagtgcaataa ctcagtttcg ggtctcctct 1140 gaactgttag gtcaaactga aaatggcttgtcctggaata ccaagagtgg tggttcacaa 1200 cacgatttgt atgtatgtcc tttgaaaaatccacctagtg atttggaaga attacaaata 1260 attgttgatg aatgtactac ccatgcgcagtttgttacta tgcgtgcagc tagcaccttc 1320 tttgttgatg ttcagctagg ctggtattggaggggttatt actatacccc acaattaagt 1380 ggttggtctt atcagatgaa aacaccagatggacagatat tctatgatct aaaaacttcg 1440 aaaatcttct ttgtccagga caaccaaaacgtgttctttc tccataataa actcaacaaa 1500 caaactggtt acagctggga ttgggtagaatggctaaaac atgacatgaa tgaggacaaa 1560 gacgaaaact ttaaatggta cttttcgcgtgatgacctta ccattccttc cgttgaaggg 1620 cttaacttcc gccacattcg ctgttacgctgacaaccagc agttaaaggt gatcataagc 1680 ggttcacgtt ggggcggttg gtactccacttacgataaag ttgaaagtaa tgtcgaagat 1740 aagattttgg tcaaagatgg ttttgatcgcttt 1773

Below are the amino acid sequences of individual clinical isolates.

JL (SEQ ID NO: 3) MPNPVRFVYR VDLRSPEEIF EHGFSTLGDV RNFFEHILST NFGRSYFISTSETPTAAIRF FGSWLREYVP EHPRRAYLYE IRADQHFYNA RATGENLLDL MRQRQVVFDSGDREMAQMGI RALRTSFAYQ REWFTDGPIA AANVRSAWLV DAVPVEPGHA HHPAGRVVETTRINEPEMHN PHYQELQTQA NDQPWLPTPG IATPVHLSIP QAASVADVSE GTSASLSFACPDWSPPSSNG ENPLDKCIAE KIDNYNLQSL PQYASSVKEL EDTPVYLRGI KTQKTFMLQADPQNNNVFLV EVNPKQKSSF PQTIFFWDVY QRICLKDLTG AQISLSLTAF TTQYAGQLKVHLSVSAVNAV NQKWKMTPQD SAITQFRVSS ELLGQTENGL FWNTKSGGSQ HDLYVCPLKNPPSDLEELQI IVDECTTHAQ FVTMRAASTF FVDVQLGWYW RGYYYTPQLS GWSYQMKTPDGQIFYDLKTS KIFFVQDNQN VFFLHNKLNK QTGYSWDWVE WLKHDMNEDK DENFKWYFSRDDLTIPSVEG LNFRHIRCYA DNQQLKVIIS GSRWGGWYST YDKVESNVED KILVKDGFDR F*RJL1 (SEQ ID NO: 4) MPNPVRFVYR VDLRSPEEIF EHGFSTLGDV RNFFEHILSTNFGRSYFIST SETPTAAIRF FGSWLREYVP EHPRRAYLYE IRADQHFYNA RATGENLLDLMRQRQVVFDS GDREMAQMGI RALRTSFAYQ REWFTDGPIA AANVRSAWLV DAVPVEPGHAHHPAGRVVET TRINEPEMHN PHYQELQTQA NDQPWLPTPG IATPVHLSIP QAASVADVSEGTSASLSFAC PDWSPPSSNG ENPLDKCIAE KIDNYNLQSL PQYASSVKEL EDTPVYLRGIKTQKTFMLQA DPQNNNVFLV EVNPKQKSSF PQTIFFWDVY QRICLKDLTG AQISLSLTAFTTQYAGQLKV HLSVSAVNAV NQKWKMTPQD SAITQFRVSS ELLGQTENGL FRNTKSGGSQHDLYVCPLKN PPSDLEELQI IVDECTTHAQ FVTMRAASTF FVDVQLGWYW RGYYYTPQLSGWSYQMKTPD GQIFYDLKTS KIFFVQDNQN VFFLHNKLNK QTGYSWDWVE WLKHDMNEDKDENFKWYFSR DDLTIPSVEG LNFRHIRCYA DNQQLKVIIS GSRWGGWYST YDKVESNVEDKILVKDGFDR F* L2 (SEQ ID NO: 5) MPNPVRFVYR VDLRSPEEIF EHGFSTLGDVRNFFEHILST NFGRSYFIST SETPTAAIRF FGSWLREYVP EHPRRAYLYE IRADQHFYNARATGENLLDL MRQRQVVFDS GDREMAQMGI RALRTSFAYQ REWFTDGPIA AANVRSAWLVDAVPVEPGHA HHPAGRVVET TRINEPEMHN PHYQELQTQA NDQPWLPTPG IATPVHLSIPQAASVADVSE GTSASLSFAC PDWSPPSSNG ENPLGKCIAE KIDNYNLQSL PQYASSVKELEDTPVYLRGI KTQKTFMLQA DPQNNNVFLV EVNPKQKSSF PQTIFFWDVY QRICLKDLTGAQISLSLTAF TTQYAGQLKV HLSVSAVNAV NQKWKMTPQD SAITQFRVSS ELLGQTENGLFWNTKSGGSQ HDLYVCPLKN PPSDLEELQI IVDECTTHAQ FVTMRAASTF FVDVQLGWYWRGYYYTPQLS GWSYQMKTPD GQIFYDLKTS KIFFVQDNQN VFFLHNKLNK QTGYSWDWVEWLKHDMNEDK DENFKWYFSR DDLTIPSVEG LNFRHIRCYA DNQQLKVIIS GSRWGGWYSTYDKVESNVED KILVKDGFDR F* S1 (SEQ ID NO: 2) MPNPVRFVYR VDLRSPEEIFEHGFSTLGDV RNFFEHIPST NFGRSYFIST SETPTAAIRF FGSWLREYVP EHPRRAYLYEIRADQHFYNA RATGENLLDL MRQRQVVFDS GDREMAQMGI RALRTSFAYQ REWFTDGPIAAANVRSAWLV DAVPVEPGHA HHPAGRVVET TRINEPEMHN PHYQELQTQA NDQPWLPTPGIATPVHLSIP QAASVADVSE GTSASLSFAC PDWSPPSSNG ENPLDKCIAE KIDNYNLQSLPQYASSVKEL EDTPVYLRGI KTQKTFMLQA DPQNNNVFLV EVNPKQKPSF PQTIFFWDVYQRICLKDLTG AQISLSLTAF TTQYAGQLKV HLSVSAVNAV NQKWKMTPQD SAITQFRVSSELLGQTENGL SWNTKSGGSQ HDLYVCPLKN PPSDLEELQI IVDECTTHAQ FVTMRAASTFFVDVQLGWYW RGYYYTPQLS GWSYQMKTPD GQIFYDLKTS KIFFVQDNQN VFFLHNKLNKQTGYSWDWVE WLKHDMNEDK DENFKWYFSR DDLTIPSVEG LNFRHIRCYA DNQQLKVIISGSRWGGWYST YDKVESNVED KILVKDGFDR F*

These sequence data are summarized below.

-   1. Translation of the nucleotide sequence of the clinical isolates    showed changes in amino acid positions at 38, 245, 308, 371, 391 and    392.-   2. All the clinical isolates have changes at amino acid position    371^(Ile→Ser).-   3. JL had only one change at aa position 371^(Ile→Ser).-   4. RJL1 had one more additional change (comparing to JL) at aa    position 392^(Trp→Arg).-   5. L2 had one more additional change (comparing to JL) at aa    position 245^(Asp→Gly).-   6. S1 had three additional changes (comparing to JL) at aa positions    38^(Leu→Pro,) 308^(Ser→Pro) and 391^(Phe→Ser).

Expression and Purification of Recombinant CARDS Protein.

DNA fragments were generated by digesting plasmid pCR-cards with NdeIand BamHI and ligated into pET19b to generate pET-cards. The plasmid wastransformed into competent E. coli BL21 (DE3) cells grown to a densityof 2×10⁹ cells/ml at 37° C. in standard LB broth containing 100 μg/mlampicillin (Sigma-Aldrich). Induction of recombinant protein synthesiswas accomplished by addition of 100 μM of isopropyl thioβ-galactopyranoside (Sigma-Aldrich), and bacteria were incubated for 3 hat 37° C. under aeration at 220 rpm. Cells from 1 ml samples werepelleted, resuspended in 250 of sample buffer (4% SDS, 125 mM Tris [pH6.8], 10% 2-ME, 10% glycerol, 0.2% bromophenol blue), and heated to 95°C. for 5 min. 10 μl aliquots of test samples were analyzed on 12%SDS/polyacrylamide gels. Recombinant colonies were screened forresistance to ampicillin and expression of a protein product of thecorrect size, and one recombinant clone from each construct was selectedfor further study. Verification of specific clones was achieved byrestriction digestion and limited DNA sequencing. Fusion proteins werepurified from recombinant E. coli under native condition by nickelaffinity chromatography using the manufacturer's protocol (Qiagen).

Preparation of Antisera Against Recombinant Mycoplasma Proteins.

Mice were immunized subcutaneously with 50-100 μg of recombinant totalCARDS protein suspended in complete Freund's adjuvant (no peptides ortruncated domains). Individual mice were boosted three times with thesame amount of recombinant antigen in incomplete Freund's adjuvant at14-day intervals. Serum samples were collected and used forimmunological characterization. Monoclonal antibodies were producedusing recombinant CARDS toxin and hybridoma supernatants were screenedfor immunoreactivity with CARDS protein and truncated peptides.

Full length recombinant CARDS Toxin (rTOX) and the amino terminal. D1domain of recombinant CARDS Toxin (rD1) were separated on 4-12%preparative gels, transferred to nitrocellulose and reacted with variousconcentrations (1:2, 1:10 and 1:50 or 1:100) of primary mouse antibodiesagainst rTOX or rD1 (Monoclonal antibodies 11D1-2H10, isotype IgGgl andmonoclonal antibody 19C4-2G10-1E1-2B9, isotype IgG3). Membranes werewashed and reacted with alkaline phosphatase-conjugated goat anti-mouseIgG. Blots were washed again, followed by color development withNBT-BCIP reagent. Both antibodies bound a protein of approximately 70kDa MW in membranes containing rTOX and both antibodies bound peptidesof 28 kDa MW and 32 kDa MW in membranes containing rD1.

Primers Designed to Express Specific Domains of CARDS

Introduced restriction sites are indicated by underline. Changes innucleotide sequences are given in bold.

ttttta c a t atgccaaatcctgtt Primer 1 (SEQ ID NO: 12)tttttacatatgccaaatcctgttag Primer 1a (SEQ ID NO: 72) gg atc ctctacgcaatgcatttgtctag 372D1R (SEQ ID NO: 65) catatgccaacaccaggaatagctactc 372D2F (SEQ ID NO: 66) ggatccactaccagcctagctgaac . . . 372D2R (SEQ ID NO: 67) c at atgggtcagctcaaagtgcaccttag 372D3F (SEQ ID NO: 68) gatcgcttttagcga gg a tcctttaacg Primer 2 (SEQ ID NO: 64) Amplified region of CARDS toxinnucleic acid encoding D1 1 (SEQ ID NO: 74) atgccaaatc ctgttagatttgtttaccgt gttgatttga gaagccctga agaaattttt  60 gaacatggct tttcaactttaggtgatgtg agaaatttct ttgaacacat tctctccact 120 aattttggta gaagctattttatttccact tcagaaacac ccacagcagc tattcgcttc 180 tttggtagct ggttacgggaatatgtacca gagcacccca gaagggctta cttatatgaa 240 attcgtgccg accaacacttttacaatgcc cgcgccactg gggagaactt gttagattta 300 atgcgtcaaa gacaagtagtatttgactct ggtgatcgag aaatggcaca aatgggaatt 360 agagctttac gcacttcctttgcgtatcaa cgtgaatggt ttaccgatgg tccaattgca 420 gcagctaatg tccgtagtgcttggctagta gatgctgttc ccgttgaacc tggtcatgct 480 caccacccgg ctggtcgtgttgtagagact actagaatta atgaaccgga aatgcacaac 540 cctcattatc aagagctgcaaacccaagcc aatgatcaac catggttgcc aacaccagga 600 atagctactc ctgtacatttatcaattccc caagcagctt ccgttgctga tgtttcggaa 660 ggtacttccg cttcgctatcgtttgcgtgc cctgattgga gtccaccttc tagtaatggt 720 gaaaatccgc tagacaaatgcattgcg 747

Domains Expected to be Expressed in E. Coli Using the Above Primers.

Overlapping amino acids within domains are indicated by underline.

Domain 1 (SEQ ID NO:69): Primer 1 and 372D1R   1MPNPVRFVYR VDLRSPEEIF EHGFSTLGDV RNFFEHILST NFGRSYFIST  51SETPTAAIRF FGSWLREYVP EHPRRAYLYE IRADQHFYNA RATGENLLDL 101MRQRQVVFDS GDREMAQMGI RALRTSFAYQ REWFTDGPIA AANVRSAWLV 151DAVPVEPGHA HHPAGRVVET TRINEPEMHN PHYQELQTQA NDQP

LPTPG 201 IATPVHLSIP QAASVADVSE GTSASLSFAC PD

SPPSSNG ENPLDKCIA Theoretical pI/Mw: 5.54/28127.37Domain 1 with His tag (underlined) (SEQ ID NO:75)MGHHHHHHHHHHSSGHTDDDDKH   1MPNPVRFVYR VDLRSPEEIF EHGFSTLGDV RNFFEHILST NFGRSYFIST  51SETPTAAIRF FGSWLREYVP EHPRRAYLYE IRADQHFYNA RATGENLLDL 101MRQRQVVFDS GDREMAQMGI RALRTSFAYQ REWFTDGPIA AANVRSAWLV 151DAVPVEPGHA HHPAGRVVET TRINEPEMHN PHYQELQTQA NDQP

LPTPG 201 IATPVHLSIP QAASVADVSE GTSASLSFAC PD

SPPSSNG ENPLDKCIA Theoretical pI/Mw with the tag: 5.95/30894.20Domain 2: (SEQ ID NO:70) 372D2F and 372D2R P

LPTPG 201 IATPVHLSIP QAASVADVSE GTSASLSFAC PD

SPPSSNG ENPLDKCIAE 251KIDNYNLQSL PQYASSVKEL EDTPVYLRGI KTQKTFMLQA DPQNNNVFLV 301EVNPKQKSSF PQTIFFWDVY QRICLKDLTG AQISLSLTAF TTQY AGQLKV 351HLSVSAVNAV NQK

KMTPQD IAITQFRVSS ELLGQTENGL F

NTKSGGSQ 401 HDLYVCPLKN PPSDLEELQI IVDECTTHAQ FVTMRAASTF FVDVQLG

Y Theoretical pI/Mw: 5.05/28378.10Domain 3 (SEQ ID NO:71): 372D3F and Primer 2 AGQLKV 351 HLSVSAVNAV NQK

WKMTPQD IAITQFRVSS ELLGQTENGL F

WNTKSGGSQ 401 HDLYVCPLKN PPSDLEELQI IVDECTTHAQ FVTMRAASTF FVDVQLGWY W451 RGYYYTPQLS GWSYQMKTPD GQIFYDLKTS KIFFVQDNQN VFFLHNKLNK 501QTGYSWDWVE WLKHDMNEDK DENFKWYFSR DDLTIPSVEG LNFRHIRCYA 551DNQQLKVIIS GSRWGGWYST YDKVESNVED KILVKDGFDR FTheoretical pI/Mw: 5.69/28966.52

Production of Recombinant N Terminal Domain of CARDS Toxin rD1

To produce rD1, the D1 PCR fragment (SEQ ID NO:74) encoding the cardsfirst 249 amino acids (SEQ ID NO:69) was cloned into the E. coliHis¹⁰-tagged expression vector, pET19b (Novagen), using NdeI and BamHIrestriction sites incorporated into the oligonucleotide primers used toamplify this nucleic acid 5′ tttttacatatgccaaatcctgttag 3′ (SEQ IDNO:72) and 5′ ggatcctctacgcaatgcatttgtctag 3′(SEQ ID NO:65). Because theNdeI site in the vector overlaps an ATG start codon, cloning the D1fragment into this site places the fragment in perfect register with thevector-derived His-tagged ribosome binding site. The amino acid sequenceof the expressed protein with the His tag is shown in SEQ ID NO:75.

After cloning the D1 PCR fragment into pET19b and confirming theidentify of the cloned fragment by DNA sequencing, a recombinant plasmidwas used to transform E. coli strain BL21 (λDE3). Transformants weregrown to mid-log phase before inducing D1 expression by addition of IPTGto a final concentration of 1 mM. After four hours, cells were harvestedby centrifugation at 8000 g for 15 minutes at 4° C. and the pellet wasresuspended in 50 mM phosphate buffer ph 8.0, containing 300 mM NaCl, 10mM imidazole and complete, EDTA-free protease inhibitor (Sigma). Cellswere disrupted by sonication; cellular debris and membranes werepelleted by centrifugation at 16000 g for 30 minutes and discarded; thesupernatant was mixed with Ni-NTA agarose slurry and left on a rocker atroom temperature for one hour; and then the slurry was loaded into acolumn. The Ni-NTA agarose packed column was extensively washed with 10mM imidazole, 20 mM imidazole, and 50 mM imidazole in the same bufferused for pellet resuspension. Finally, D1 was purified in a single stepelution with 250 mM imidazole in the same buffer. Fractions containingpurified protein were desalted using P10 columns (Amersham Biosciences)with TG buffer (20 mM Tris-Cl, pH 7.4, 5% glycerol) and concentratedusing YM-10 (Amicon) membranes. Protein concentrations were estimatedusing a BCA protein assay kit (Pierce) and the protein was aliquoted andstored at −80° C.

Cytopathology in Chinese Hamster Ovary (CHO) Cells

Cells were seeded into 25 cm² cell culture flasks and incubation wascontinued until monolayer confluence was achieved. Then, recombinantCARDS protein (20 μg/ml or 40 μg/ml) was added for 24 hours. Monolayerswere photographed on an Olympus CK40 microscope at 20× magnification.

In CHO cells, the recombinant toxin causes cytopathology with anassociated “foamy” appearance, rounding of cells and cell detachmentfrom monolayers.

ADP-Ribosylation of G Proteins Following Incubation of CARDS Proteinwith HEp-2 Cells.

Confluent HEp-2 cells were incubated with medium alone or in thepresence of 40 μg/ml CARDS protein for 16 hours at 37° C. Cells werewashed and incubated with fresh medium at 37° C. for four hours. Cellfree extracts (CFE) were prepared and assayed for ADP-ribosylation (CFEwere incubated with 40 μg/ml CARDS protein for one hour with 0.1 μM[³²P]NAD in 100 mM Tris pH 7.5, 20 mM DTT). The reaction mixture wasprecipitated with 10% TCA and proteins were resolved in a 4-15% gradientgel by SDS-PAGE and transferred to nitrocellulose membrane forautoradiography. As shown in the autoradiogram in FIG. 2, the CARDSexotoxin exhibits ADP ribosylating activity.

Animal Model of M. pneumoniae Infection

Sera from mice infected with M. pneumoniae has been shown to seroconvertto the CARDS toxin. These mice will be used as an animal model tofurther assess the role of the CARDS Toxin in infection and diseaseprogression.

Although the present process has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims.

Throughout this application, various patents and non-patent publicationsare referenced. The disclosures of these patents and publications intheir entireties are hereby incorporated by reference into thisapplication in order to more fully describe the state of the art towhich this invention pertains.

1. An isolated polypeptide comprising an amino acid sequence selectedfrom the group consisting of the amino acid sequence of SEQ ID NO:2, theamino acid sequence of SEQ ID NO:3, the amino acid sequence of SEQ IDNO:4, the amino acid sequence of SEQ ID NO:5, and the amino acidsequence of SEQ ID NO:6. 2-8. (canceled)
 9. A composition comprising thepolypeptide of claim 1 and a pharmaceutically acceptable carrier. 10-45.(canceled)